Modified proteins, designer toxins, and methods of making thereof

ABSTRACT

The present invention concerns methods of reducing the antigenicity of a proteinaceous compound while maintaining the compounds biological activity, as well as proteinaceous compositions with biological activity but reduced antigenicity. These methods and compositions have significant benefits to a subject in need of such compounds and compositions. Also included are modified toxin compounds that are truncated and/or possess reduce antigenicity. Such designer toxins have therapeutic, diagnostic, and preventative benefits, particularly as immunotoxins. Methods of treating cancer using these immunotoxins are provided.

The present application is a divisional application of U.S. Ser. No.10/074,596 filed Feb. 12, 2002, now U.S. Pat. No. 7,083,957, and claimsthe benefit of priority to U.S. Provisional Patent Application No.60/268,402, filed on Feb. 12, 2001, which is incorporated by referencein its entirety herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of molecularbiology and toxicology. More particularly, it concerns methods ofgenerating modified proteins that are shorter and/or less antigenicpolypeptides, as well as compositions comprising such polypeptides.Shorter and less antigenic versions of the plant toxin gelonin aredescribed herein. Such modified proteins have therapeutic and diagnosticuses, for example, as immunotoxins.

2. Description of Related Art

Peptides, polypeptides, and proteins have numerous preventative,diagnostic, and therapeutic benefits. One disadvantage, however, is thatsuch proteinaceous compounds may elicit an immune response to thecompounds in the subject who hopes to receive their benefit. An immuneresponse to the compounds can reduce, or altogether eliminate, thebenefits that can be achieved through their use. Thus, it is a generaldesire to decrease the antigenicity or immunogenicity of a compoundwhose efficacy may be reduced by its eliciting an immune response in thehost.

One specific type of protein, monoclonal antibodies, have been the focusof much research and development for preventative, diagnostic, andtherapeutic benefits. Highly specific immunotoxins recognizing a varietyof cell-surface antigens have been developed and tested over the lasttwo decades. The attractive feature of immunotoxins is that these potentagents require very few molecules to be successfully delivered to thecorrect intracellular compartment in order to elicit a cytotoxic effect.Immunotoxins have been constructed containing various toxins such assaponin, abrin, ricin A chain (RTA), pseudomonas exotoxin (PE),diptheria toxin (DT), and gelonin.

Problems associated with the in vivo use of immunotoxins generallyinclude: vascular damage leading to a capillary leak syndrome,mistargeting due to recognition of the toxin portion by thereticuloendothelial system, heterogeneity of target antigen expression,and development of anti-toxin antibodies leading to a narrowed therapywindow of approximately 14 days. The development of anti-toxin andanti-conjugate antibodies may also prevent retreatment of patientsdespite evidence of antitumor effect. Prolonged use of immunotoxins inpatients has provoked problems as well. Immunoconjugates containing RTAand PE have been found to be highly immunogenic in patients. Inaddition, the size of these proteins in immunotoxin constructs(approximately 30 kDa) is suspected to prevent effective penetration ofimmunoconjugates into solid tumors, The structural modification of TypeI proteins such as RTA has, for the most part, been unsuccessful(Munishkin et al., 1995). Numerous RTA mutants modifying several aminoacids have been generated. In 1995, Wool et al. described 45 singleamino acids deletions of RTA. Of those, only 8 single amino aciddeletions were shown to have biological activity although the relativethe relative biological activities of these deletion mutants compared tonative RTA have not been examined. While interesting, the studiesexamining RTA are of limited value since, for example, RTA has only 30%sequence homology with other toxins such as gelonin.

Specific applications of monoclonal antibody (MAb)-based procedures havetraditionally been found in the diagnosis and therapy of human cancers.However, clinical use of these agents has met with limited success dueto drawbacks associated with this approach, e.g. heterogeneity ofantigen expression, poor tumor penetration into solid tumors due in partto antibody size, and antigenicity of the antibodies (Roselli et al.,1993; Berkower, 1996; Pullybland et al., 1997; Panchagnula et al., 1997;Panchal, 1998). To circumvent these problems, a number of molecularapproaches have been applied to reconfigure the conventional antibodystructure into mouse:human chimeras, completely human antibodies orreshaped antibody fragments containing the antigen-binding portions ofthe original structure in a smaller and simpler (single-chain) format(Bird et al., 1988; Kipriyanov et al., 1994; Owens et al., 1994;McCartney et al., 1995; Worn et al., 1998). Single-chain antibodies(scfv, sfv), retaining the binding characteristics of the parentimmunoglobulin (IgG), consist of the antibody V_(L) and V_(H) domainslinked by a designed flexible peptide linker (Wels et al., 1992; Kuruczet al., 1993). Furthermore, scFvs may be preferred in clinical anddiagnostic applications currently involving conventional MAbs or Fabfragments thereof, since their smaller size may allow better penetrationof tumor tissue, improved pharmacokinetics, and a reduction in theimmunogenicity observed with intravenously administered murineantibodies.

Among the few target antigens that are expressed at high levels inmelanoma cells compared to normal tissue is the surface domain of a highmolecular weight glycoprotein (gp240) found on a majority of melanomacell lines and fresh tumor samples (Kantor et al., 1982). Two murineantibodies (designated 9.2.27 and ZME-018) recognizing differentepitopes on this antigen have been previously isolated and described(Morgan et al., 1981; Wilson et al., 1981). The murine monoclonalantibody ZME-018 possesses high specificity for melanoma and isminimally reactive with a variety of normal tissues, making it apromisingcandidate for further study. Clinical trials examining theability of this antibody to localize within melanoma lesions havedemonstrated selective concentration in metastatic tumors (Macey et al.,1988; Koizumi et al., 1988).

Successful development of tumor-targeted therapeutic agents isdependent, in part, on the site-specific delivery of therapeutic agentsand also on the biological activity of the delivered agent. Monoclonalantibodies have been employed to impart selectivity to otherwiseindiscriminately cytotoxic agents such as toxins, radionuclides, andgrowth factors (Williams et al., 1990; Rowlinson-Busza et al., 1992;Wahl, 1994). One such molecule is gelonin, a 29-kDaribosome-inactivating plant toxin with a potency and mechanism of actionsimilar to ricin A-chain (RTA) but with improved stability and reducedtoxicity (Stirpe et al., 1992; Rosenblum et al., 1995). Previous studiesin our lab have identified and examined the biological properties ofnumerous chemical conjugates of the plant toxin gelonin and variousantibodies (Boyle et al., 1995; Xu et al., 1996; Rosenblum et al.,1999). In previous studies, antibody ZME-018 was chemically coupled topurified gelonin, and this immunoconjugate demonstrated specificcytotoxicity against antigen-positive melanoma cells both in tissueculture and in human tumor xenograft models (Rosenblum et al., 1991;Mujoo et al., 1995). However, this construct, like immunotoxinsgenerally, has inherent problems of antigenicity in human patients.

Given the side effects of immunotoxins and the limited progress made inreducing these problems, there is a continued need for the developmentof less antigenic proteins, polypeptides, and peptides for use in thetreatment, prevention, and diagnosis of diseases and conditions.Replacement of antigenic sequences in the toxin molecule is a conceptwith respect to non-antibody polypeptides, such as toxins. While thisconcept has been used successfully with replacement of murineimmunoglobulin framework domains with those of human immunoglobinframework domains creating a human/mouse chimeric molecule, the sameconcept has never been successfully applied to other moleculesparticularly toxins or enzymes from plant sources, or by using themethods described herein.

SUMMARY OF THE INVENTION

The present invention concerns methods of creating and preparingproteinaceous compounds that are modified to form a modified proteinthat possesses an advantage over a non-modified or native protein. Thepresent invention also includes compositions that are generated fromthese methods.

In some embodiments of the invention, a recombinant gelonin toxin isprovided that is altered with respect to the native gelonin sequence.The recombinant gelonin toxin may have amino acids replaced or removedas compared to the native gelonin protein sequence (shown in SEQ IDNO:1), which is disclosed in U.S. Pat. No. 5,631,348, which is hereinincorporated by reference and which is provided by GenBank accessionnumber L12243. The recombinant gelonin toxin or the present inventiondoes not have all of the amino acids of SEQ ID NO:1, but in someembodiments, comprises a core toxin region defined as amino acidresidues 110-210 of SEQ ID NO:1. Other compounds of the presentinvention include a recombinant gelonin toxin that contains the coretoxin region in addition to having at least 10, 20, 30, 40, 50, 60, 70,80, 90, 100 or more contiguous amino acid residues of SEQ ID NO:1 inaddition to the core toxin region. It is contemplated that compounds ofthe present invention also include multiple regions that includecontiguous amino acid residues of SEQ ID NO:1. For example, a compoundmay include the core toxin region in addition to 10 contiguous aminoacid residues of SEQ ID NO:1 before the core toxin region and 20contiguous amino acid residues of SEQ ID NO:1 after the core toxinregion.

A recombinant gelonin toxin of the invention also includes a gelonintoxin that is truncated with respect to the native sequence, such thatthe toxin is lacking at least 5, 10, 20, 30, 40, 50, or more amino acidsof SEQ ID NO:1. In some embodiments of the invention, the toxin containsthe core toxin region, but is missing amino acids anywhere outside thecore toxin region. In addition to deletions, the recombinant gelonintoxin of the invention may have an amino acid in place of a removedamino acid. For example, the glycine residue at position 7 in thegelonin protein sequence may be replaced with a non-glycine amino acidresidue or a modified amino acid. If the glycine residue at position 7is merely removed, the alanine at position 8 in SEQ ID NO:1 becomesposition 7, but is not considered a replacement because the positions ofthe amino acids are simply shifted by 1 position. It is contemplatedthat at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, or more amino acids may be replaced.

In further embodiments of the present invention, a recombinant gelonintoxin may be attached to a second polypeptide. In some instances, thesecond polypeptide serves to target the gelonin toxin to a particularcell type (including cells having a particular genotype or phenotype,such as a cancer cell or a cell infected with a pathogen), part of thebody, or other specific location. Proteinaceous compounds of theinvention, therefore, include a compound that contains both arecombinant gelonin toxin, such as a modified gelonin toxin and a secondpolypeptide. In some embodiments, the two polypeptides are conjugated toone another, while in other embodiments the polypeptides are engineeredrecombinantly to produce a fusion protein. Conjugated compounds may beattached to one another by a linker. It is contemplated that modifiedproteins of the present invention may include additional polypeptidecompositions, all or some of which may be covalently linked to oneanother.

The present invention concerns multipolypeptide compositions in whichmore than one polypeptide entity is presented as a single compound.Thus, a modified protein may be attached to a second, third, fourth,fifth, sixth or more polypeptides. Alternatively, two or more modifiedproteins may be presented as a. singly proteinaceous compound. In someembodiments of the invention, the second polypeptide is an antibody,such as an antibody with an antigen binding region. It is contemplatedthat an antibody may be directed against a tumor antigen, an oncogeneproduct, a cellular receptor, or any other compound that localizes themultipolypeptide composition. As disclosed herein, the secondpolypeptide may be an enzyme, a cytokine, a cytotoxic molecule, a growthfactor, a ligand or receptor, or any molecule that is capable ofmodifying cell growth characteristics.

Other compositions of the invention include a modified enzyme producedby a process that includes: a) identifying one or more antigenic regionsin the enzyme using an antibody; b) removing one or more antigenicregions from the enzyme to form a modified enzyme; and c) determiningthat the modified enzyme has enzymatic activity. An enzyme is abiological entity that catalyzes a specific chemical reaction in a cell;it may be a protein or a nucleic acid molecule. However, it iscontemplated that any methods discussed with respect to enzymes may beapplied to polypeptides generally. An antigenic region is a region of apolypeptide that is specifically recognized by an antibody or T-cellreceptor of a particular organism. It is understood that a region may beantigenic in one species but not in another species, and therefore,antigenicity of a compound is a characteristic that is relative to aparticular organism. In addition to removing amino acids that are partof an antigenic region, it is contemplated that amino acids from morethan one antigenic region may be removed from an enzyme of the presentinvention. Amino acids from all orpart of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more antigenic regions may beremoved from the polypeptide. In some cases, the removed region isreplaced with a region that is less antigenic than the removed region.Of course, it is understood that amino acids flanking an antigenicregion may also be removed, for example, for purposes of convenience.Thus, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, or more amino acids flanking one or both sides of an antigenicregion may be removed or replaced.

A less antigenic region or regions may be identified by searching aprotein database search for regions that are homologous to or have someresidues in common with an antigenic region. An antigenic region may beidentified, and this sequence is used to identify known proteinsequences of the organism in which less antigenicity with respect to amodified protein is desirable. Thus, a human protein database may beemployed to find human protein sequences that have multiple residuesthat are identical or comparable to residues of an antigenic region ofprotein desired to be less antigenic in humans. A residue is comparableto another residue if they are not identical but they share similarchemical properties. Such relationships are well known to those of skillin the art.

In some embodiments, an antibody is employed to identify an antigenicregion. It is contemplated that an antibody may be polyclonal. Theorganism source of the antibody is the same species of organism in whichthe modified protein is desired to be less antigenic. Therefore, if anenzyme or protein is desired to be less antigenic in a human, it isdesirable in some embodiments that human antibodies be used either toidentify an antigenic region or to determine whether a modified proteinis less antigenic than a non-modified protein (native or recombinantfull-length). In preferred embodiments, a modified enzyme or protein isevaluated for reduced or lower antigenicity by comparing theantigenicity of a modified enzyme or protein with an unmodified enzymeor protein; this can be accomplished by i) obtaining a sample from asubject prior to exposure to or administration of a modified protein andusing the sample to compare the antigenicity of the modified protein andthe unmodified version of the same protein, or ii) obtaining a samplefrom a subject after exposure to or administration of a modified proteinand using the sample to compare the antigenicity of the modified proteinand the unmodified version of the same protein. A sample may be anycomposition that contains antibodies or immune cells, including bodilyfluids such as blood (serum). The sample may then be used to implementan immunodetection method, such as an ELISA. It is contemplated that thesubject may be naive with respect to the unmodified protein, though itis preferable that a subject providing the sample have been previouslyexposed to the unmodified protein. In some embodiments it may beappropriate that a sample is culture media from a monoclonal antibodyhybridoma.

While in other aspects of the invention, determining whether modifiedprotein or enzyme possesses activity may be accomplished by assaying themodified compound for activity, such as enzymatic activity.

Any enzyme may be modified according to methods of the presentinvention. The enzyme may be a hydrolase (e.g., deaminase, esterase,glycosidase, lipase, nuclease, peptidase, phosphatase,phosphodiesterase, and proteinase); isomerase (e.g., epimerase, mutase,and racemase); ligase or synthetase (e.g., acyl-CoA synthetase,amino-acyl-tRNA synthetase, and carboxylase); lyase (e.g., aldolase,decarboxylase, dehydratase, and nucleotide cyclase); oxidoreductase(e.g., dehydrogenase, dioxygenase, hydrogenase, monooxygenase,nitrogenase, oxidase, and reductase); and transferase (e.g.,acyltransferase, aminotransferase, glycosyltransferase, kinase,methyltransferase, nucleotidyltransferase, phosphorylase, andsulphotransferase). In specific embodiments, the enzyme is classified asa toxin, which means it is toxic to a cell, tissue, or organism.Specifically contemplated as part of the invention are toxins producedby plants, such as gelonin. As previously discussed a modified enzyme,like modified gelonin polypeptides of the invention, may be attached toadditional polypeptides. It is understood that any of the embodimentswith respect to modified gelonin may be applied to modified enzymes, andvice versa.

The present invention also concerns methods of generating modifiedproteins that have reduced antigenicity, and in some cases, particularlywith respect to a subject. In some embodiments, the method includes: a)selecting a protein one desires to administer to a first subject; b)identifying a region of the protein that is antigenic in the firstsubject using antiserum from either the first subject or a secondsubject of the same species as the first subject; c) generating amodified protein in which the identified region is absent; and d)confirming the modified protein has reduced antigenicity. As previouslydiscussed, this last step may be accomplished using a sample, such asserum, from an individual who has been previously exposed to theunmodified version of the modified protein or from the individual inwhich a reduced immune response against the modified protein is desired.

It is further contemplated that methods of generating a modified proteininclude steps of screening a human protein database to identify a lessantigenic region that has homology to the antigenic region of theprotein and replacing the antigenic region with all or part of theidentified region that is less antigenic to form a modified protein.Screening of a large human protein database is not required but isdesirable. Thus, if the sequence of a particular human protein that hashomology or identical residues with an antigenic region is knownindependently from screening a human protein database, this method wouldbe included in the scope of the present invention. For example, one mayknow the sequence of the human homolog of a mouse enzyme whose reducedantigenicity is desired; replacing regions in the mouse protein withresidues from the human sequence concerns the present invention. Methodsand compositions of the invention involve replacing, deleting, and/ormodifying amino acid residues of a polypeptide. A residue that isreplaced renders both the order and number of the remaining amino acidsthe same as the polypeptide before the residue was replaced. A residuemay be replaced with a conservative or non-conservative residue. Aresidue that is deleted does not disturb the order of the remainingamino acids, but reduces the number of residues of the polypeptide byone. A residue that is modified is one that is chemically altered; thischange does not alter the order or number of remaining amino acids inthe polypeptide.

In some embodiments, methods involve using recombinant nucleic acidtechnology to achieve a modified protein or enzyme. Thus, a cDNAsequence for enzyme desired to be modified may be manipulated such thata nucleic acid sequence that encodes an antigenic region is replacedwith a nucleic acid sequence that encodes a less antigenic region.Alternatively, a modified protein may be generated by removing theidentified region. A region that is removed is considered absent. Anabsent region may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100 or more amino acid residues. Moreover, amodified protein may have more than one antigenic region removed orreplaced, and amino acids flanking the region may also be removed orreplaced. It is contemplated that the absent antigenic region may bereplaced with the same number of amino acid residues that are removed.

In the methods of the present invention, an antigenic region may beidentified or a modified protein may be evaluated using an ELISA assay.A subject may be a mammal, such as a human.

Other compositions of the invention include a humanized recombinantgelonin toxin having at least 3 amino acids from one or more ofantigenic domains 1, 2, 3, or 4 replaced with amino acids less antigenicin a human than a recombinant gelonin toxin with the replaced aminoacids. Antigenic domains of a gelonin toxin are described elsewhere. Itis contemplated that at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, 100, or moreamino acids from antigenic domains 1, 2, 3, and/or 4 are replaced,deleted, or modified. Amino acids from at least 2, 3, or 4 antigenicdomains may be manipulated.

Additional embodiments of the invention provide a recombinant gelonintoxin produced by a process involving: a) identifying at least oneregion in a gelonin toxin that is antigenic in a mammal; and b)replacing at least a portion of the antigenic region with a region lessantigenic in the mammal. It is contemplated that gelonin toxin may berecombinant, that is, derived from a nucleic acid sequence that has beenmanipulated in vitro. The process may also include comparing theidentified antigenic region with mammalian amino acid sequences, wherebya region less antigenic in the mammal is identified or identifying aregion that is less antigenic in the mammal. In some embodiments, themammal is a human. As previously mentioned, any of the methods andcompositions disclosed herein may be applied to any other methods andcompositions described herein.

The present invention also concerns methods of treatment using thecompositions of the invention. They may used in the treatment of anydisease in which treatment takes the form of killing or eliminatingcertain cells or organisms, which is effected by toxins of theinvention. It is contemplated that embodiments discussed with respect toone composition or method may be applied to any other composition ormethod of the invention.

In some embodiments, there is a method of killing cancer or tumor cellsby providing to the cells an effective amount of an immunotoxin thatincludes all or part of a gelonin toxin, such as its core toxin region,and all or part of an antibody, which is employed to direct theimmunotoxin to a particular cell. An “effective amount” refers to anamount that achieves the intended goal. In the case of a method forkilling a cancer or tumor cell, it is the amount to achieve the killingof a cancer or tumor cell. Other methods of the invention includemethods for treating cancer in a patient by administering to the patientan effective amount of a composition comprising an immunotoxincomprising a core toxin region of gelonin and single chain antibody thatspecifically targets a cancer cell. An “effective amount” with respectto treatment refers to conferring a therapeutic benefit on the subject.The term “therapeutic benefit” used throughout this application refersto anything that promotes or enhances the well-being of the subject withrespect to the medical treatment of his condition. In the context ofcancer (though it may apply to other conditions as well), therapeuticbenefit, which includes treatment of pre-cancer, cancer, andhyperproliferative diseases, includes the following nonexhaustiveexamples: extension of the subject's life by any period of time,decrease or delay in the neoplastic development of the disease, decreasein hyperproliferation, reduction in tumor growth, delay of metastases,reduction in cancer cell or tumor cell proliferation rate, and adecrease in pain to the subject that can be attributed to the subject'scondition.

In some embodiments of the invention the toxin is gelonin. In stillfurther embodiments, the immunotoxin includes all or part of the aminoacid sequence of SEQ ID NO:1. It is contemplated that the immunotoxinmay include fewer amino acids than the full-length gelonin proteinsequence, though it includes the full-length sequence in someembodiments. It is further contemplated that the toxin may be humanizedand it may be any of the toxins or constructs disclosed or describedherein.

In further embodiments the antibody of the immunotoxin is humanizedand/or is a single chain antibody. In methods of the invention, anantibody targets the immunotoxin to the targeted cancer cell, though itmay not be full-length, so long as it allows for specific targeting. Insome embodiments, the antibody (which includes antibody fragments)specifically targets (i.e., binds) an antigen on the surface of thetargeted cell. In more specific embodiments, the antibody targets atumor antigen. The antibody can be any mammalian antibody, though it isspecifically contemplated that the antibody is a mouse, rabbit, rat,goat, or monkey antibody. The antibody, though from a different species,may be humanized according to the invention or other methods known tothose of ordinary skill in the art. In cases in which the antibody is asingle chain antibody, it may include 9.2.27 or ZME-018, which areantibodies directed to melanoma cells. In specific examples, theimmunotoxin is scfvMEL-2018 or scfvMEL-2025 (SEQ ID NO:11), describedherein.

The cancer cell that is targeted may be a cell from prostate, lung,brain, skin, liver, breast, lymphoid, stomach, testicular, ovarian,pancreas, bone, bone marrow, head and neck, cervical, esophagus, eye,gall bladder, kidney, adrenal glands, heart, colon, or blood.Alternatively, the cancer patient may have a cancer in or from theorgans/tissue identified above. In some embodiments of the invention,the cancer cell is a melanoma cell. It is contemplated that the canceror tumor cell may be in a patient. In some embodiments, the patient willbe administered an effective amount of a therapeutic composition, whichrefers to the amount needed to achieve a particular desired result, suchas treatment. In the context of cancer, for example, the desired resultmay be killing of a cancer or tumor cell.

The immunotoxin may be included in a pharmaceutically orpharmacologically acceptable composition. As part of a treatmentregimen, a patient may also receive other anti-cancer therapy, such aschemotherapy, radiotherapy, gene therapy, surgery, or otherimmunotherapy.

In even further embodiments, it is contemplated that the immunotoxin maybe provided to a cell or a patient by providing an expression constructthat contains a nucleic acid sequence encoding the immunotoxin and iscapable of expressing the immunotoxin. In some embodiments, theexpression construct is a viral vector, including, but not limited to,an adenovirus vector, an adeno-associated virus vector, a hepatitisvirus, a herpesvirus, a lentivirus, a retrovirus, or a vaccinia virus.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. ELISA done with anti-rGelonin antibody against human serumsamples.

FIG. 2. Epitopes of rGelonin recognized by human anti-geloninantibodies.

FIG. 3A-3B. Gelonin Deletion Constructs. The structures of gelonindeletion constructs are shown.

FIG. 4. Schematic of PCR-based construction of the sfvMEL/rGel fusiontoxin and ligation into pET-32a derived vectors.

FIG. 5 Complete DNA sequence analysis of the sfvMEL/rGel fusionconstruct (SEQ ID NO:10).

FIG. 6 Comparative binding of the parental ZME-rGel chemical conjugateand sfvMEL-rGel fusion construct (same as “sfvMEL/rGel). Binding toA-375 cells was assessed using ELISA and a polyclonal rabbitanti-gelonin polyclonal antibody. The binding of both constructs totarget cells was similar although slightly higher binding was observedfor the recombinant fusion construct.

FIG. 7 Comparative in vitro cytotoxicity of the parental ZME-rGelchemical conjugate and sfvMEL-rGel fusion construct on antigen-positiveA375 human melanoma cells. Cells were plated and then treated for 72 hwith various doses of sfvMEL/rGel fusion construct, ZME-rGel chemicalconjugate or free recombinant gelonin. IC₅₀ values for bothimmunoconjugates were approximately 8 nM, while the IC₅₀ for therecombinant gelonin was several orders of magnitude higher atapproximately 2×10³ nM.

FIG. 8 Competitive inhibition of sfvMEL/rGel immunotoxin with ZMEantibody. Various concentrations of the recombinant immunotoxin wereadded to A-375 human melanoma cells in log-phase culture inquadruplicate. To another set of wells, a fixed concentration ofantibody ZME (50 υg/ml) was admixed with various doses of sfvMEL/rGelimmunotoxin and incubated for 72 h. Addition of free ZME antibodyresulted in approximately a 3-fold reduction in immunotoxincytotoxicity.

FIG. 9 Nude mice bearing well-developed melanoma tumors (A-375) growingin the right flank were treated (i.v.) with either saline (controls) orsfvMEL/rGel at 2 mg/kg or 20 mg/kg (total dose) for 4 consecutive days(arrows). Tumor areas were measured for 30 days. The saline-treatedcontrol tumors increased from 30 to 150 mm² over this period. Tumorstreated with the lowest immunotoxin dose increased from 30 to 60 mm².Animals treated with the highest immunotoxin dose showed no overallincrease in tumor size from the original 30 mm².

FIG. 10 The cytotoxicity of scfvMEL-CFR2018 (also known as“sfvMEL-CFR2018) was compared with the cytotoxicity of scfvMEL-CFR2025on A375-M melanoma cells in an in vitro cytotoxicity assay.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Proteins and polypeptides with reduced antigenicity can providetremendous benefits as compositions administered to an organism with animmune system. Methods of designing and producing such proteins andpolypeptides are described herein, as are the resultant molecules.Enzymes are particularly interesting candidates for these methodsbecause it may be desirable to preserve the enzymatic activity of aparticular enzyme, but also reduce its antigenicity in a subject thatmay benefit from the protein's enzymatic activity. Ribosome-inactivatingproteins (RIPs) are an example of such a protein. Thus, in someembodiments of this invention, nucleic acid and polypeptide compositionsare provided that involve plant toxins, such as gelonin. Proteins may bedesigned to provide the toxic function of one polypeptide in acombination with another polypeptide, such as a targetting molecule.These designer toxins have a wide variety of applications.

I. Proteinaceous Compounds

In certain embodiments, the present invention concerns novelcompositions comprising a proteinaceous molecule that has been modifiedrelative to a native or wild-type protein. In some embodiments thatproteinaceous compound has been deleted of amino acid residues; in otherembodiments, amino acid residues of the proteinaceous compound have beenreplaced, while in still further embodiments both deletions andreplacements of amino acid residues in the proteinaceous compound havebeen made. Furthermore, a proteinaceous compound may include an aminoacid molecule comprising more than one polypeptide entity. As usedherein, a “proteinaceous molecule,” “proteinaceous composition,”“proteinaceous compound,” “proteinaceous chain” or “proteinaceousmaterial” generally refers, but is not limited to, a protein of greaterthan about 200 amino acids or the full length endogenous sequencetranslated from a gene; a polypeptide of greater than about 100 aminoacids; and/or a peptide of from about 3 to about 100 amino acids. Allthe “proteinaceous” terms described above may be used interchangeablyherein. Furthermore, these terms may be applied to fusion proteins orprotein conjugates as well.

In certain embodiments the size of the at least one proteinaceousmolecule may comprise, but is not limited to, about or at least 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 or greateramino molecule residues, and any range derivable therein. Compounds ofthe invention may include the above-mentioned number of contiguous aminoacids from SEQ ID NO:1 and/or SEQ ID NO:11. It is contemplated thatembodiments with respect to SEQ ID NO:1 may be employed with respect toany other amino acid sequences described herein, including SEQ ID NO:11,and vice versa, if appropriate.

Accordingly, the term “proteinaceous composition” encompasses aminomolecule sequences comprising at least one of the 20 common amino acidsin naturally synthesized proteins, or at least one modified or unusualamino acid, including but not limited to those shown on Table 1A below.

TABLE 1A Modified and Unusual Amino Acids Abbr. Amino Acid Aad2-Aminoadipic acid Baad 3-Aminoadipic acid Bala β-alanine,β-Amino-propionic acid Abu 2-Aminobutyric acid 4Abu 4-Aminobutyric acid,piperidinic acid Acp 6-Aminocaproic acid Ahe 2-Aminoheptanoic acid Aib2-Aminoisobutyric acid Baib 3-Aminoisobutyric acid Apm 2-Aminopimelicacid Dbu 2,4-Diaminobutyric acid Des Desmosine Dpm 2,2′-Diaminopimelicacid Dpr 2,3-Diaminopropionic acid EtGly N-Ethylglycine EtAsnN-Ethylasparagine Hyl Hydroxylysine AHyl allo-Hydroxylysine 3Hyp3-Hydroxyproline 4Hyp 4-Hydroxyproline Ide Isodesmosine AIleallo-Isoleucine MeGly N-Methylglycine, sarcosine MeIleN-Methylisoleucine MeLys 6-N-Methyllysine MeVal N-Methylvaline NvaNorvaline Nle Norleucine Orn Ornithine

As used herein, an “amino molecule” refers to any amino acid, amino acidderivative or amino acid mimic as would be known to one of ordinaryskill in the art. In certain embodiments, the residues of theproteinaceous molecule are sequential, without any non-amino moleculeinterrupting the sequence of amino molecule residues. In otherembodiments, the sequence may comprise one or more non-amino moleculemoieties. In particular embodiments, the sequence of residues of theproteinaceous molecule may be interrupted by one or more non-aminomolecule moieties.

1. Functional Aspects

The present invention concerns modified proteins, particularly thosethat confer a therapeutic benefit to a subject because the modifiedprotein exhibits a functional activity that is comparable to theunmodified protein, yet the modified protein possesses an additionaladvantage in the subject over the unmodified protein, such as havingless antigenicity and/or eliciting fewer side effects, and/or havingbetter or longer efficacy. Thus, when the present application refers tothe function or activity of “modified protein” one of ordinary skill inthe art would understand that this includes, for example, a proteinthat 1) performs the same activity or has the same specificity as theunmodified protein, but that may have a different level of activity; and2) possesses an additional advantage over the unmodified protein.Determination of activity may be achieved using assays familiar to thoseof skill in the art, particularly with respect to the protein'sactivity, and may include for comparison purposes, for example, the useof native and/or recombinant versions of either the modified orunmodified protein.

2. Modified Proteins

Modified proteins of the present invention may possess deletions and/orsubstitutions of amino acids; thus, a protein with a deletion, a proteinwith a substitution, and a protein with a deletion and a substitutionare modified proteins. In some embodiments these modified proteins mayfurther include insertions or added amino acids, such as with fusionproteins or proteins with linkers, for example. A “modified deletedprotein” lacks one or more residues of the native protein, but possessesthe specificity and/or activity of the native protein. A “modifieddeleted protein” may also have reduced immunogenicity or antigenicity.An example of a modified deleted protein is one that has an amino acidresidue deleted from at least one antigenic region-that is, a region ofthe protein determined to be antigenic in a particular organism, such asthe type of organism that may be administered the modified protein.

Substitutional or replacement variants typically contain the exchange ofone amino acid for another at one or more sites within the protein andmay be designed to modulate one or more properties of the polypeptide,particularly to reduce its immunogenicity/antigenicity, reduce any sideeffects in a subject, or increase its efficacy. Substitutions of thiskind preferably are conservative, that is, one amino acid is replacedwith one of similar shape and charge. Conservative substitutions arewell known in the art and include, for example, the changes of: alanineto serine; arginine to lysine; asparagine to glutamine or histidine;aspartate to glutamate; cysteine to serine; glutamine to asparagine;glutamate to aspartate; glycine to proline; histidine to asparagine orglutamine; isoleucine to leucine or valine; leucine to valine orisoleucine; lysine to arginine; methionine to leucine or isoleucine;phenylalanine to tyrosine, leucine or methionine; serine to threonine;threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan orphenylalanine; and valine to isoleucine or leucine. An antigenic regionof a polypeptide may be substituted for a less antigenic region; theless antigenic region may contain residues that are identical to thecorresponding residues in the native protein, yet also contain someconservative substitutions and/or nonconservative substitutions.

In addition to a deletion or substitution, a modified protein maypossess an insertion of residues, which typically involves the additionof at least one residue in the polypeptide. This may include theinsertion of a targeting peptide or polypeptide or simply a singleresidue. Terminal additions, called fusion proteins, are discussedbelow.

The term “biologically functional equivalent” is well understood in theart and is further defined in detail herein. Accordingly, sequences thathave between about 70% and about 80%, or between about 81% and about90%, or even between about 91% and about 99% of amino acids that areidentical or functionally equivalent to the amino acids of a nativepolypeptide are included, provided the biological activity of theprotein is maintained. A modified protein may be biologicallyfunctionally equivalent to its native counterpart.

It also will be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

The following is a discussion based upon changing of the amino acids ofa protein to create an equivalent, or even an improved,second-generation molecule. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, binding sites to substrate molecules. Since it is theinteractive capacity and nature of a protein that defines that protein'sbiological functional activity, certain amino acid substitutions can bemade in a protein sequence, and in its underlying DNA coding sequence,and nevertheless produce a protein with like properties. It is thuscontemplated by the inventors that various changes may be made in theDNA sequences of genes without appreciable loss of their biologicalutility or activity, as discussed below. Table 1 shows the codons thatencode particular amino acids. A proteinaceous molecule has “homology”or is considered “homologous” to a second proteinaceous molecule if oneof the following “homology criteria” is met: 1) at least 30% of theproteinaceous molecule has sequence identity at the same positions withthe second proteinaceous molecule; 2) there is some sequence identity atthe same positions with the second proteinaceous molecule and at thenonidentical residues, at least 30% of them are conservativedifferences, as described herein, with respect to the secondproteinaceous molecule; or 3) at least 30% of the proteinaceous moleculehas sequence identity with the second proteinaceous molecule, but withpossible gaps of nonidentical residues between identical residues. Asused herein, the term “homologous” may equally apply to a region of aproteinaceous molecule, instead of the entire molecule. If the term“homology” or “homologous” is qualified by a number, for example, “50%homology” or “50% homologous,” then the homology criteria, with respectto 1), 2), and 3), is adjusted from “at least 30%” to “at least 50%.”Thus it is contemplated that there may homology of at least 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or morebetween two proteinaceous molecules or portions of proteinaceousmolecules.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte & Doolittle, 1982). It is accepted that therelative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine(−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still produce a biologicallyequivalent and immunologically equivalent protein. In such changes, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions generally are based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take into consideration the variousforegoing characteristics are well known to those of skill in the artand include: arginine and lysine; glutamate and aspartate; serine andthreonine; glutamine and asparagine; and valine, leucine and isoleucine.

Table 2 provides a list of proteins and polypeptides that may bemodified according to the methods of the present invention describedherein. Non-human polypeptides are specifically contemplated as targetsof the methods of the invention to reduce their antigenicity in a human.It is contemplated that non-human proteins with therapeutic value arewithin the scope of the invention. Any other proteins or polypeptidesdiscussed in the specification may be modified according to methods ofthe present invention.

TABLE 2 Protein Protein Genus Subgenus Protein Species ProteinSubspecies 1) Toxins Ribosome Inhibitory Proteins Gelonin Ricin A ChainPseudomonas Exotoxin Diptheria Toxin Mitogillin Saporin 2)Cytokines/Growth Interleukins IL-1, IL-2, IL-3, IL- Factors 4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL- 11, IL-12, IL-13, IL- 14, IL-15,IL-16, IL- 17, IL-18, IL-19 TNF LT Interferons IFNα, IFNβ, IFNγ ColonyGM-CSF, G-CSF, M- Stimulating CSF, CSF Factors LIF Fibroblast bFGF, FGF,FGF-1, Growth Factors FGF-2, FGF-3, FGF- 4, FGF-8, FGF-9, FGF-10,FGF-18, FGF-20, FGF, 23 VEGF 3) Enzymes Oxidoreductases TransferasesTransferring one- Methyltransferases carbon groups Carboxyl andcarbamoyltransferases Amidinotransferases Transferring aldehyde orketone residues Acyltransferases Acyltransferases AminoacyltransferasesGlycosyltransferases Hexosyltransferases Transferring alkyl or arylgroups, other than methyl groups Transferring Transaminases nitrogenousgroups Oximinotransferases Transferring Phosphotransferases phosphorous-containing groups Diphosphotransferases NucleotidyltransferasesTransferring sulfur- Sulfur-transferases containing groupsSulfotransferases CoA-transferases Transferring selenium-containinggroups Hydrolases Acting on ester bonds Glycosylases Acting on etherbonds Acting on peptide bonds (peptide hydrolases) Acting on carbon-nitrogen bonds, other than peptide bonds Acting on acid anhydridesActing on carbon- carbon bonds. Acting on halide bonds Acting onphosphorus-nitrogen bonds. Acting on sulfur- nitrogen bonds Acting oncarbon- phosphorus bonds Acting on sulfur- sulfur bonds LyasesCarbon-carbon lyases. Carbon-oxygen lyases Carbon-nitrogen lyasesCarbon-sulfur lyases Carbon-halide lyases Phosphorus-oxygen lyasesIsomerases Racemases and epimerases Cis-trans-isomerases Intramolecularoxidoreductases Intromolecular transferases (mutases)Phosphotransferases (phosphomutases) Ligases Forming carbon- oxygenbonds Forming carbon- sulfur bonds Forming carbon- nitrogen bonds.Forming carbon- carbon bonds Forming phosphoric ester bonds

Another embodiment for the preparation of modified polypeptidesaccording to the invention is the use of peptide mimetics. Mimetics arepeptide-containing molecules that mimic elements of protein secondarystructure. See, e.g., Johnson (1993). The underlying rationale behindthe use of peptide mimetics is that the peptide backbone of proteinsexists chiefly to orient amino acid side chains in such a way as tofacilitate molecular interactions, such as those of antibody andantigen. A peptide mimetic is expected to permit molecular interactionssimilar to the natural molecule. These principles may be used, inconjunction with the principles outline above, to engineer secondgeneration modified protein molecules having many of the naturalproperties of a native protein, but with altered and, in some cases,even improved characteristics.

3. Multipolypeptide Proteinaceous Compounds

The present invention concerns a proteinaceous compound that may includeamino acid sequences from more than one polypeptide. A proteinaceouscompound or molecule, for example, could include a modified toxin withan antigen binding region of an antibody. The multipolypeptideproteinaceous molecule may be two or more proteins chemically conjugatedto one another or it may be a fusion protein of two or more polypeptidesencoded by the same nucleic acid molecule. A fusion or conjugatedprotein comprising a toxin and a second polypeptide with activity may bereferred to as a “dual toxin.” Thus, a multipolypeptide proteinaceouscompound may be comprised of all or part of a first polypeptide and allor part of a second polypeptide, a third polypeptide, a fourthpolypeptide, a fifth polypeptide, a sixth polypeptide, a seventhpolypeptide, an eight polypeptide, a ninth polypeptide, a tenthpolypeptide, or more polypeptides.

Designer toxins themselves in general, have no capability to bind to thecell surface or internalize within specific cells. Therefore, theseagents require either chemical conjugation to or fusion withagents/proteins which are capable of binding to specific target cellsand internalizing into the cell efficiently once bound. Table 3 providesa list of proteins and polypeptides that may be conjugated or fused totoxins of the present invention, particularly in embodiments involvingtargeting the engineered proteinaceous compounds to a particular placed,such as specific cell types or parts of the body. The invention furtherincludes adjoining all or part of a toxin molecule to all or part of anyof the proteins listed in Table 2. It is contemplated that the inventionincludes, but is not limited to, the examples provided in these Tables 2and 3.

TABLE 3 Genus Subgenus Species Subspecies 1) Antibodies PolyclonalMonoclonal non-recombinant recombinant chimeric single chain diabodymultimeric 2) Cytokines/ Interleukins IL-1, IL-2, IL-3, IL- Lymphokines/4, IL-5, IL-6, IL-7, Growth Factors IL-8, IL-9, IL-10, IL-11, IL-12,IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19 EGF Colony GM-CSF,G-CSF, Stimulating M-CSF Factors (CSF) 3) Small Chemical Nicotine ThatBind Cell Surface and Are Internalized ATP Amino Acids Dopamine

a. Fusion Proteins

A specialized kind of insertional variant is the fusion protein. Thismolecule generally has all or a substantial portion of the nativemolecule, linked at the N- or C-terminus, to all or a portion of asecond polypeptide. For example, fusions typically employ leadersequences from other species to permit the recombinant expression of aprotein in a heterologous host. Another useful fusion includes theaddition of an immunologically active domain, such as an antibodyepitope or other tag, to facilitate targeting or purification of thefusion protein. The use of 6×His and GST (glutathione S transferase) astags is well known. Inclusion of a cleavage site at or near the fusionjunction will facilitate removal of the extraneous polypeptide afterpurification. Other useful fusions include linking of functionaldomains, such as active sites from enzymes such as a hydrolase,glycosylation domains, cellular targeting signals or transmembraneregions.

Immunotoxins are specifically contemplated as an embodiment of thepresent invention. An immunotoxin is a cytotoxic compound comprising atleast a portion of an antibody and a portion of a toxin molecule. Theantibody and the toxin may be fused or conjugated to each other. Moredetail about immunotoxins is provided infra.

b. Conjugated Proteins

The present invention further provides conjugated polypeptides, such astranslated proteins, polypeptides and peptides, generally of themonoclonal type, that are linked to at least one agent to form anantibody conjugate. In order to increase the efficacy of antibodymolecules as diagnostic or therapeutic agents, it is conventional tolink or covalently bind or complex at least one desired molecule ormoiety. Such a molecule or moiety may be, but is not limited to, atleast one effector or reporter molecule. Effector molecules comprisemolecules having a desired activity, e.g., cytotoxic activity.Non-limiting examples of effector molecules which have been attached toantibodies include toxins, anti-tumor agents, therapeutic enzymes,radio-labeled nucleotides, antiviral agents, chelating agents,cytokines, growth factors, and oligo- or poly-nucleotides. By contrast,a reporter molecule is defined as any moiety that may be detected usingan assay. Non-limiting examples of reporter molecules which have beenconjugated to antibodies include enzymes, radiolabels, haptens,fluorescent labels, phosphorescent molecules, chemiluminescentmolecules, chromophores, luminescent molecules, photoaffinity molecules,colored particles or ligands, such as biotin.

Any antibody of sufficient selectivity, specificity or affinity may beemployed as the basis for an antibody conjugate. Such properties may beevaluated using conventional immunological screening methodology knownto those of skill in the art. Sites for binding to biological activemolecules in the antibody molecule, in addition to the canonical antigenbinding sites, include sites that reside in the variable domain that canbind pathogens, B-cell superantigens, the T cell co-receptor CD4 and theHIV-1 envelope (Sasso et al., 1989; Shorki et al., 1991; Silvermann etal., 1995; Cleary et al., 1994; Lenert et al., 1990; Berberian et al.,1993; Kreier et al., 1991). In addition, the variable domain is involvedin antibody self-binding (Kang et al., 1988), and contains epitopes(idiotopes) recognized by anti-antibodies (Kohler et al., 1989).

Certain examples of antibody conjugates are those conjugates in whichthe antibody is linked to a detectable label. “Detectable labels” arecompounds and/or elements that can be detected due to their specificfunctional properties, and/or chemical characteristics, the use of whichallows the antibody to which they are attached to be detected, and/orfurther quantified if desired. Another such example is the formation ofa conjugate comprising an antibody linked to a cytotoxic oranti-cellular agent, and may be termed “immunotoxins.”

Antibody conjugates may be employed for use as diagnostic agents.Antibody diagnostics generally fall within two classes, those for use inin vitro diagnostics, such as in a variety of immunoassays, and/or thosefor use in vivo diagnostic protocols, generally known as“antibody-directed imaging”.

Many appropriate imaging agents are known in the art, as are methods fortheir attachment to antibodies (see, for e.g., U.S. Pat. Nos. 5,021,236;4,938,948; and 4,472,509, each incorporated herein by reference). Theimaging moieties used can be paramagnetic ions; radioactive isotopes;fluorochromes; NMR-detectable substances; X-ray imaging.

In the case of paramagnetic ions, one might mention by way of exampleions such as chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III), with gadoliniumbeing particularly preferred. Ions useful in other contexts, such asX-ray imaging, include but are not limited to lanthanum (III), gold(III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnosticapplication, one might mention astatine²¹¹, ¹⁴carbon, ⁵¹chromium,³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen,iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus,rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur, technicium^(99m) and/oryttrium⁹⁰. ¹²⁵I is often being preferred for use in certain embodiments,and technicium^(99m) and/or indium¹¹¹ are also often preferred due totheir low energy and suitability for long range detection. Radioactivelylabeled monoclonal antibodies of the present invention may be producedaccording to well-known methods in the art. For instance, monoclonalantibodies can be iodinated by contact with sodium and/or potassiumiodide and a chemical oxidizing agent such as sodium hypochlorite, or anenzymatic oxidizing agent, such as lactoperoxidase. Monoclonalantibodies according to the invention may be labeled withtechnetium^(99m) by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the antibody to this column.Alternatively, direct labeling techniques may be used, e.g., byincubating pertechnate, a reducing agent such as SNCl₂, a buffersolution such as sodium-potassium phthalate solution, and the antibody.Intermediary functional groups which are often used to bindradioisotopes which exist as metallic ions to antibody arediethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetraceticacid (EDTA).

Among the fluorescent labels contemplated for use as conjugates includeAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, RhodamineRed, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed.

Another type of antibody conjugates contemplated in the presentinvention are those intended primarily for use in vitro, where theantibody is linked to a secondary binding ligand and/or to an enzyme (anenzyme tag) that will generate a colored product upon contact with achromogenic substrate. Examples of suitable enzymes include urease,alkaline phosphatase, (horseradish) hydrogen peroxidase or glucoseoxidase. Preferred secondary binding ligands are biotin and/or avidinand streptavidin compounds. The use of such labels is well known tothose of skill in the art and are described, for example, in U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149and 4,366,241; each incorporated herein by reference.

Yet another known method of site-specific attachment of molecules toantibodies comprises the reaction of antibodies with hapten-basedaffinity labels. Essentially, hapten-based affinity labels react withamino acids in the antigen binding site, thereby destroying this siteand blocking specific antigen reaction. However, this may not beadvantageous since it results in loss of antigen binding by the antibodyconjugate.

Molecules containing azido groups may also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter & Haley, 1983). Inparticular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; and Dholakia et al., 1989) and may be used asantibody binding agents.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody(U.S. Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein byreference). Monoclonal antibodies may also be reacted with an enzyme inthe presence of a coupling agent such as glutaraldehyde or periodate.Conjugates with fluorescein markers are prepared in the presence ofthese coupling agents or by reaction with an isothiocyanate. In U.S.Pat. No. 4,938,948, imaging of breast tumors is achieved usingmonoclonal antibodies and the detectable imaging moieties are bound tothe antibody using linkers such as methyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

In other embodiments, derivatization of immunoglobulins by selectivelyintroducing sulfhydryl groups in the Fc region of an immunoglobulin,using reaction conditions that do not alter the antibody combining siteare contemplated. Antibody conjugates produced according to thismethodology are disclosed to exhibit improved longevity, specificity andsensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).Site-specific attachment of effector or reporter molecules, wherein thereporter or effector molecule is conjugated to a carbohydrate residue inthe Fc region have also been disclosed in the literature (O'Shannessy etal., 1987). This approach has been reported to produce diagnosticallyand therapeutically promising antibodies which are currently in clinicalevaluation.

i. Linkers/Coupling Agents

Multiple peptides or polypeptides, such as with a conjugatedimmunotoxin, may be joined via a biologically-releasable bond, such as aselectively-cleavable linker or amino acid sequence. For example,peptide linkers that include a cleavage site for an enzymepreferentially located or active within a tumor environment arecontemplated. Exemplary forms of such peptide linkers are those that arecleaved by urokinase, plasmin, thrombin, Factor IXa, Factor Xa, or ametallaproteinase, such as collagenase, gelatinase, or stromelysin.Alternatively, peptides or polypeptides may be joined to an adjuvant.

Amino acids such as selectively-cleavable linkers, synthetic linkers, orother amino acid sequences may be used to separate proteinaceousmoieties. Additionally, while numerous types of disulfide-bondcontaining linkers are known that can successfully be employed toconjugate the toxin moiety with the targeting agent, certain linkerswill generally be preferred over other linkers, based on differingpharmacologic characteristics and capabilities. For example, linkersthat contain a disulfide bond that is sterically “hindered” are to bepreferred, due to their greater stability in vivo, thus preventingrelease of the toxin moiety prior to binding at the site of action.Furthermore, certain advantages in accordance with the invention will berealized through the use of any of a number of toxin moieties, includinggelonin and a deglycosylated A chain of ricin.

It can be considered as a general guideline that any biochemicalcross-linker that is appropriate for use in an immunotoxin will also beof use in the present context, and additional linkers may also beconsidered.

Cross-linking reagents are used to form molecular bridges that tietogether functional groups of two different molecules, e.g., astablizing and coagulating agent. To link two different proteins in astep-wise manner, hetero-bifunctional cross-linkers can be used thateliminate unwanted homopolymer formation.

It is contemplated that cross-linkers may be implemented with themodified protein molecules of the invention. Bifunctional cross-linkingreagents have been extensively used for a variety of purposes includingpreparation of affinity matrices, modification and stabilization ofdiverse structures, identification of binding sites, and structuralstudies. In the context of the invention, such cross-linker may be usedto stabilize the polypeptide or to render it more useful as atherapeutic, for example, by improving the modified protein's targetingcapability or overall efficacy. Cross-linkers may also be cleavable,such as disulfides, acid-sensitive linkers, and others. Homobifunctionalreagents that carry two identical functional groups proved to be highlyefficient in inducing cross-linking between identical and differentmacromolecules or subunits of a macromolecule, and linking ofpolypeptides to specific binding sites on binding partners.Heterobifunctional reagents contain two different functional groups. Bytaking advantage of the differential reactivities of the two differentfunctional groups, cross-linking can be controlled both selectively andsequentially. The bifunctional cross-linking reagents can be dividedaccording to the specificity of their functional groups, e.g., amino,sulfhydryl, guanidino, indole, carboxyl specific groups. Of these,reagents directed to free amino groups have become especially popularbecause of their commercial availability, ease of synthesis and the mildreaction conditions under which they can be applied. A majority ofheterobifunctional cross-linking reagents contains a primaryamine-reactive group and a thiol-reactive group.

Exemplary methods for cross-linking ligands to liposomes are describedin U.S. Pat. No. 5,603,872 and U.S. Pat. No. 5,401,511, eachspecifically incorporated herein by reference in its entirety). Variousligands can be covalently bound to liposomal surfaces through thecross-linking of amine residues. Liposomes, in particular, multilamellarvesicles (MLV) or unilamellar vesicles such as microemulsified liposomes(MEL) and large unilamellar liposomes (LUVET), each containingphosphatidylethanolamine (PE), have been prepared by establishedprocedures. The inclusion of PE in the liposome provides an activefunctional residue, a primary amine, on the liposomal surface forcross-linking purposes. Ligands such as epidermal growth factor (EGF)have been successfully linked with PE-liposomes. Ligands are boundcovalently to discrete sites on the liposome surfaces. The number andsurface density of these sites will be dictated by the liposomeformulation and the liposome type. The liposomal surfaces may also havesites for non-covalent association. To form covalent conjugates ofligands and liposomes, cross-linking reagents have been studied foreffectiveness and biocompatibility. Cross-linking reagents includeglutaraldehyde (GAD), bifunctional oxirane (OXR), ethylene glycoldiglycidyl ether (EGDE), and a water soluble carbodiimide, preferably1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Through the complexchemistry of cross-linking, linkage of the amine residues of therecognizing substance and liposomes is established.

In another example, heterobifunctional cross-linking reagents andmethods of using the cross-linking reagents are described (U.S. Pat. No.5,889,155, specifically incorporated herein by reference in itsentirety). The cross-linking reagents combine a nucleophilic hydrazideresidue with an electrophilic maleimide residue, allowing coupling inone example, of aldehydes to free thiols. The cross-linking reagent canbe modified to cross-link various functional groups and is thus usefulfor cross-linking polypeptides and sugars. Table 3 details certainhetero-bifunctional cross-linkers considered useful in the presentinvention.

TABLE 4 HETERO-BIFUNCTIONAL CROSS-LINKERS Spacer Arm Length\after LinkerReactive Toward Advantages and Applications cross-linking SMPT Primaryamines Greater stability 11.2 A Sulfhydryls SPDP Primary aminesThiolation  6.8 A Sulfhydryls Cleavable cross-linking LC-SPDP Primaryamines Extended spacer arm 15.6 A Sulfhydryls Sulfo-LC-SPDP Primaryamines Extended spacer arm 15.6 A Sulfhydryls Water-soluble SMCC Primaryamines Stable maleimide reactive group 11.6 A SulfhydrylsEnzyme-antibody conjugation Hapten-carrier protein conjugationSulfo-SMCC Primary amines Stable maleimide reactive group 11.6 ASulfhydryls Water-soluble Enzyme-antibody conjugation MBS Primary aminesEnzyme-antibody conjugation  9.9 A Sulfhydryls Hapten-carrier proteinconjugation Sulfo-MBS Primary amines Water-soluble  9.9 A SulfhydrylsSIAB Primary amines Enzyme-antibody conjugation 10.6 A SulfhydrylsSulfo-SIAB Primary amines Water-soluble 10.6 A Sulfhydryls SMPB Primaryamines Extended spacer arm 14.5 A Sulfhydryls Enzyme-antibodyconjugation Sulfo-SMPB Primary amines Extended spacer arm 14.5 ASulfhydryls Water-soluble EDC/Sulfo-NHS Primary amines Hapten-Carrierconjugation 0 Carboxyl groups ABH Carbohydrates Reacts with sugar groups11.9 A Nonselective

In instances where a particular polypeptide, such as gelonin, does notcontain a residue amenable for a given cross-linking reagent in itsnative sequence, conservative genetic or synthetic amino acid changes inthe primary sequence can be utilized.

4. Protein Purification

While some of the embodiments of the invention involve recombinantproteins, the invention concerns also methods and processes forpurifying proteins, including modified proteins and recombinantproteins. Generally, these techniques involve, at one level, the crudefractionation of the cellular milieu to polypeptide and non-polypeptidefractions. Having separated the polypeptide from other proteins, thepolypeptide of interest may be further purified using chromatographicand electrophoretic techniques to achieve partial or completepurification (or purification to homogeneity). Analytical methodsparticularly suited to the preparation of a pure peptide areion-exchange chromatography, exclusion chromatography; polyacrylamidegel electrophoresis; isoelectric focusing. A particularly efficientmethod of purifying peptides is fast protein liquid chromatography oreven HPLC. In addition, the conditions under which such techniques areexecuted may be affect characteristics, such as functional activity, ofthe purified molecules.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide. The term “purified protein or peptide” as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the protein or peptide is purified to any degreerelative to its naturally-obtainable state. A purified protein orpeptide therefore also refers to a protein or peptide, free from theenvironment in which it may naturally occur. A “substantially purified”protein or peptide.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, about 96%, about 97%, about 98%, about 99%, about 99.2%,about 99.4%, about 99.6%, about 99.8%, about 99.9% or more of theproteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

The use of a peptide tag in combination with the methods andcompositions of the invention is also contemplated. A tag takesadvantage of an interaction between two polypeptides. A portion of oneof the polypeptides that is involved in the interaction may used as atag. For instance, the binding region of glutathione S transferase (GST)may be used as a tag such that glutathione beads can be used to enrichfor a compound containing the GST tag. An epitope tag, which an aminoacid region recognized by an antibody or T cell receptor, may be used.The tag may be encoded by a nucleic acid segment that is operativelylinked to a nucleic acid segment encoding a modified protein such that afusion protein is encoded by the nucleic acid molecule. Other suitablefusion proteins are those with β-galactosidase, ubiquitin, hexahistidine(6×His), or the like.

5. Antibodies

In certain embodiments, the present invention involves antibodies. Forexample, all or part of a monoclonal, single chain, or humanizedantibody may be chemically conjugated or recombinantly fused to anotherproteinaceous compound such as a modified gelonin toxin. Alternatively,other aspects of the invention involve recognizing an immune response,that is, an antibody response, to a particular antigen or antigenicregion in order to design and/or prepare a proteinaceous compound withless immunogenicity than a native form of the proteinaceous compound. Asdetailed above, in addition to antibodies generated against full lengthproteins, antibodies also may be generated in response to smallerconstructs comprising epitopic core regions, including wild-type andmutant epitopes. An epitope is an antigenic determinant. An antigen isany substance that is specifically recognized by an antibody or T-cellreceptor. An immunogen is an antigen that induces a specific immuneresponse.

As used herein, the term “antibody” is intended to refer broadly to anyimmunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally,IgG and/or IgM are preferred because they are the most common antibodiesin the physiological situation and because they are most easily made ina laboratory setting.

Monoclonal antibodies (mAbs) are recognized to have certain advantages,e.g., reproducibility and large-scale production, and their use isgenerally preferred. The invention thus provides monoclonal antibodiesof the human, murine, monkey, rat, hamster, rabbit and even chickenorigin.

The term “antibody” is used to refer to any antibody-like molecule thathas an antigen binding region, and includes antibody fragments such asFab′, Fab, F(ab′)₂, single domain antibodies (DABs), Fv, scFv (singlechain Fv), and the like. The techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (See, e.g., Harlow and Lane, “Antibodies: A Laboratory Manual,” ColdSpring Harbor Laboratory, 1988; incorporated herein by reference).

The methods for generating monoclonal antibodies (mAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody may be prepared by immunizing an animalwith an immunogenic polypeptide composition in accordance with thepresent invention and collecting antisera from that immunized animal.Alternatively, in some embodiments of the present invention, serum iscollected from persons who may have been exposed to a particularantigen. Exposure to a particular antigen may occur a work environment,such that those persons have been occupationally exposed to a particularantigen and have developed polyclonal antibodies to a peptide,polypeptide, or protein. In some embodiments of the invention polyclonalserum from occupationally exposed persons is used to identify antigenicregions in the gelonin toxin through the use of immunodetection methods.

A wide range of animal species can be used for the production ofantisera. Typically the animal used for production of antisera is arabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because ofthe relatively large blood volume of rabbits, a rabbit is a preferredchoice for production of polyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin also canbe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Suitablemolecule adjuvants include all acceptable immunostimulatory compounds,such as cytokines, toxins or synthetic compositions.

Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12,γ-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such asthur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A(MPL). RIBI, which contains three components extracted from bacteria,MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2%squalene/Tween 80 emulsion also is contemplated. MHC antigens may evenbe used. Exemplary, often preferred adjuvants include complete Freund'sadjuvant (a non-specific stimulator of the immune response containingkilled Mycobacterium tuberculosis), incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

In addition to adjuvants, it may be desirable to coadminister biologicresponse modifiers (BRM), which have been shown to upregulate T cellimmunity or downregulate suppressor cell activity. Such BRMs include,but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA);low-dose Cyclophosphamide (CYP; 300 mg/m²) (Johnson/Mead, NJ), cytokinessuch as γ-interferon, IL-2, or IL-12 or genes encoding proteins involvedin immune helper functions, such as B-7.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization.

A second, booster injection also may be given. The process of boostingand titering is repeated until a suitable titer is achieved. When adesired level of immunogenicity is obtained, the immunized animal can bebled and the serum isolated and stored, and/or the animal can be used togenerate mAbs.

mAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified polypeptide, peptide or domain, be it a wild-type ormutant composition. The immunizing composition is administered in amanner effective to stimulate antibody producing cells.

mAbs may be further purified, if desired, using filtration,centrifugation and various chromatographic methods such as HPLC oraffinity chromatography. Fragments of the monoclonal antibodies of theinvention can be obtained from the monoclonal antibodies so produced bymethods which include digestion with enzymes, such as pepsin or papain,and/or by cleavage of disulfide bonds by chemical reduction.Alternatively, monoclonal antibody fragments encompassed by the presentinvention can be synthesized using an automated peptide synthesizer.

It also is contemplated that a molecular cloning approach may be used togenerate mAbs. For this, combinatorial immunoglobulin phagemid librariesare prepared from RNA isolated from the spleen of the immunized animal,and phagemids expressing appropriate antibodies are selected by panningusing cells expressing the antigen and control cells. The advantages ofthis approach over conventional hybridoma techniques are thatapproximately 10⁴ times as many antibodies can be produced and screenedin a single round, and that new specificities are generated by H and Lchain combination which further increases the chance of findingappropriate antibodies.

Humanized monoclonal antibodies are antibodies of animal origin thathave been modified using genetic engineering techniques to replaceconstant region and/or variable region framework sequences with humansequences, while retaining the original antigen specificity. Suchantibodies are commonly derived from rodent antibodies with specificityagainst human antigens. Such antibodies are generally useful for in vivotherapeutic applications. This strategy reduces the host response to theforeign antibody and allows selection of the human effector functions.

“Humanized” antibodies are also contemplated, as are chimeric antibodiesfrom mouse, rat, or other species, bearing human constant and/orvariable region domains, bispecific antibodies, recombinant andengineered antibodies and fragments thereof. The techniques forproducing humanized immunoglobulins are well known to those of skill inthe art. For example U.S. Pat. No. 5,693,762 discloses methods forproducing, and compositions of, humanized immunoglobulins having one ormore complementarity determining regions (CDR's). When combined into anintact antibody, the humanized immunoglobulins are substantiallynon-immunogenic in humans and retain substantially the same affinity asthe donor immunoglobulin to the antigen, such as a protein or othercompound containing an epitope. Examples of other teachings in this areainclude U.S. Pat. Nos. 6,054,297; 5,861,155; and 6,020,192, allspecifically incorporated by reference. Methods for the development ofantibodies that are “custom-tailored” to the patient's disease arelikewise known and such custom-tailored antibodies are alsocontemplated.

6. Immunodetection Methods

As discussed, in some embodiments, the present invention concernsimmunodetection methods for binding, purifying, removing, quantifyingand/or otherwise detecting biological components such as antigenicregions on polypeptides and peptides. The immunodetection methods of thepresent invention can be used to identify antigenic regions of apeptide, polypeptide, or protein that has therapeutic implications,particularly in reducing the immunogenicity or antigenicity of thepeptide, polypeptide, or protein in a target subject.

Immunodetection methods include enzyme linked immunosorbent assay(ELISA), radioimmunoassay (RIA), immunoradiometric assay,fluoroimrnmunoassay, chemiluminescent assay, bioluminescent assay, andWestern blot, though several others are well known to those of ordinaryskill. The steps of various useful immunodetection methods have beendescribed in the scientific literature, such as, e.g., Doolittle M H andBen-Zeev O, 1999; Gulbis B et al., 1993; De Jager R et al., 1993; andNakamura et al., 1987, each incorporated herein by reference.

In general, the immunobinding methods include obtaining a samplesuspected of containing a protein, polypeptide and/or peptide, andcontacting the sample with a first antibody, monoclonal or polyclonal,in accordance with the present invention, as the case may be, underconditions effective to allow the formation of immunocomplexes.

These methods include methods for purifying a protein, polypeptideand/or peptide from organelle, cell, tissue or organism's samples. Inthese instances, the antibody removes the antigenic protein, polypeptideand/or peptide component from a sample. The antibody will preferably belinked to a solid support, such as in the form of a column matrix, andthe sample suspected of containing the protein, polypeptide and/orpeptide antigenic component will be applied to the immobilized antibody.The unwanted components will be washed from the column, leaving theantigen immunocomplexed to the immobilized antibody to be eluted.

The immunobinding methods also include methods for detecting andquantifying the amount of an antigen component in a sample and thedetection and quantification of any immune complexes formed during thebinding process. Here, one would obtain a sample suspected of containingan antigen or antigenic domain, and contact the sample with an antibodyagainst the antigen or antigenic domain, and then detect and quantifythe amount of immune complexes formed under the specific conditions.

In terms of antigen detection, the biological sample analyzed may be anysample that is suspected of containing an antigen or antigenic domain,such as, for example, a tissue section or specimen, a homogenized tissueextract, a cell, an organelle, separated and/or purified forms of any ofthe above antigen-containing compositions, or even any biological fluidthat comes into contact with the cell or tissue, including blood and/orserum.

Contacting the chosen biological sample with the antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, any antigenspresent. After this time, the sample-antibody composition, such as atissue section, ELISA plate, dot blot or western blot, will generally bewashed to remove any non-specifically bound antibody species, allowingonly those antibodies specifically bound within the primary immunecomplexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art.

The antibody employed in the detection may itself be linked to adetectable label, wherein one would then simply detect this label,thereby allowing the amount of the primary immune complexes in thecomposition to be determined. Alternatively, the first antibody thatbecomes bound within the primary immune complexes may be detected bymeans of a second binding ligand that has binding affinity for theantibody. In these cases, the second binding ligand may be linked to adetectable label. The second binding ligand is itself often an antibody,which may thus be termed a “secondary” antibody. The primary immunecomplexes are contacted with the labeled, secondary binding ligand, orantibody, under effective conditions and for a period of time sufficientto allow the formation of secondary immune complexes. The secondaryimmune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the antibody is used to form secondary immunecomplexes, as described above. After washing, the secondary immunecomplexes are contacted with a third binding ligand or antibody that hasbinding affinity for the second antibody, again under effectiveconditions and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system may provide forsignal amplification if this is desired.

One method of immunodetection designed by Charles Cantor uses twodifferent antibodies. A first step biotinylated, monoclonal orpolyclonal antibody is used to detect the target antigen(s), and asecond step antibody is then used to detect the biotin attached to thecomplexed biotin. In that method the sample to be tested is firstincubated in a solution containing the first step antibody. If thetarget antigen is present, some of the antibody binds to the antigen toform a biotinylated antibody/antigen complex. The antibody/antigencomplex is then amplified by incubation in successive solutions ofstreptavidin (or avidin), biotinylated DNA, and/or complementarybiotinylated DNA, with each step adding additional biotin sites to theantibody/antigen complex. The amplification steps are repeated until asuitable level of amplification is achieved, at which point the sampleis incubated in a solution containing the second step antibody againstbiotin. This second step antibody is labeled, as for example with anenzyme that can be used to detect the presence of the antibody/antigencomplex by histoenzymology using a chromogen substrate. With suitableamplification, a conjugate can be produced which is macroscopicallyvisible.

Another known method of immunodetection takes advantage of theimmuno-PCR (Polymerase Chain Reaction) methodology. The PCR method issimilar to the Cantor method up to the incubation with biotinylated DNA,however, instead of using multiple rounds of streptavidin andbiotinylated DNA incubation, the DNA/biotin/streptavidin/antibodycomplex is washed out with a low pH or high salt buffer that releasesthe antibody. The resulting wash solution is then used to carry out aPCR reaction with suitable primers with appropriate controls. At leastin theory, the enormous amplification capability and specificity of PCRcan be utilized to detect a single antigen molecule.

a. ELISAs

As detailed above, immunoassays, in their most simple and/or directsense, are binding assays. Certain preferred immunoassays are thevarious types of enzyme linked immunosorbent assays (ELISAs) and/orradioimmunoassays (RIA) known in the art. Immunohistochemical detectionusing tissue sections is also particularly useful. However, it will bereadily appreciated that detection is not limited to such techniques,and/or western blotting, dot blotting, FACS analyses, and/or the likemay also be used.

In one exemplary ELISA, antibodies are immobilized onto a selectedsurface exhibiting protein affinity, such as a well in a polystyrenemicrotiter plate. Then, a test composition suspected of containing theantigen, such as a clinical sample, is added to the wells. After bindingand/or washing to remove non-specifically bound immune complexes, thebound antigen may be detected. Detection is generally achieved by theaddition of another antibody that is linked to a detectable label. Thistype of ELISA is a simple “sandwich ELISA.” Detection may also beachieved by the addition of a second antibody, followed by the additionof a third antibody that has binding affinity for the second antibody,with the third antibody being linked to a detectable label.

In another exemplary ELISA, the samples suspected of containing theantigen are immobilized onto the well surface and/or then contacted withantibodies. After binding and/or washing to remove non-specificallybound immune complexes, the bound anti-antibodies are detected. Wherethe initial antibodies are linked to a detectable label, the immunecomplexes may be detected directly. Again, the immune complexes may bedetected using a second antibody that has binding affinity for the firstantibody, with the second antibody being linked to a detectable label.

Another ELISA in which the antigens are immobilized, involves the use ofantibody competition in the detection. In this ELISA, labeled antibodiesagainst an antigen are added to the wells, allowed to bind, and/ordetected by means of their label. The amount of an antigen in an unknownsample is then determined by mixing the sample with the labeledantibodies against the antigen during incubation with coated wells. Thepresence of an antigen in the sample acts to reduce the amount ofantibody against the antigen available for binding to the well and thusreduces the ultimate signal. This is also appropriate for detectingantibodies against an antigen in an unknown sample, where the unlabeledantibodies bind to the antigen-coated wells and also reduces the amountof antigen available to bind the labeled antibodies.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below.

In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period of hours. The wellsof the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein or solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the biological sample to betested under conditions effective to allow immune complex(antigen/antibody) formation. Detection of the immune complex thenrequires a labeled secondary binding ligand or antibody, and a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or a third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and/or antibodies with solutions such as BSA, bovine gammaglobulin (BGG) or phosphate buffered saline (PBS)/Tween. These addedagents also tend to assist in the reduction of nonspecific background.

The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 25° C. to 27° C., or maybe overnight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. An example of a washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. This may be an enzyme that willgenerate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact orincubate the first and second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immune complex formation (e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea, or bromocresolpurple, or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS),or H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generated, e.g., usinga visible spectra spectrophotometer.

b. Immunohistochemistry

The antibodies of the present invention may also be used in conjunctionwith both fresh-frozen and/or formalin-fixed, paraffin-embedded tissueblocks prepared for study by immunohistochemistry (IHC). For example,immunohistochemistry may be utilized to evaluate a particularimmunotoxin of the present invention. The method of preparing tissueblocks from these particulate specimens has been successfully used inprevious IHC studies of various prognostic factors, and/or is well knownto those of skill in the art (Brown el al., 1990; Abbondanzo et al.,1990; Allred et al., 1990).

Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen“pulverized” tissue at room temperature in phosphate buffered saline(PBS) in small plastic capsules; pelleting the particles bycentrifugation; resuspending them in a viscous embedding medium (OCT);inverting the capsule and/or pelleting again by centrifugation;snap-freezing in −70° C. isopentane; cutting the plastic capsule and/orremoving the frozen cylinder of tissue; securing the tissue cylinder ona cryostat microtome chuck; and/or cutting 25-50 serial sections.

Permanent-sections may be prepared by a similar method involvingrehydration of the 50 mg sample in a plastic microfuge tube; pelleting;resuspending in 10% formalin for 4 hours fixation; washing/pelleting;resuspending in warm 2.5% agar; pelleting; cooling in ice water toharden the agar; removing the tissue/agar block from the tube;infiltrating and/or embedding the block in paraffin; and/or cutting upto 50 serial permanent sections.

II. Nucleic Acid Molecules

A. Polynucleotides Encoding Native Proteins or Modifled Proteins

The present invention concerns polynucleotides, isolatable from cells,that are free from total genomic DNA and that are capable of expressingall or part of a protein or polypeptide. The polynucleotide may encode anative protein that may be manipulated to encode a modified protein.Alternatively, the polynucleotide may encode a modified protein, or itmay encode a polynucleotide that will be used to make a fusion proteinwith a modified protein. For example, a polynucleotide may encodemultiple moieties such as a modified gelonin polypeptide that iscovalently attached to a targeting polypeptide, e.g., a tumor antigen.It is contemplated that a single polynucleotide molecule may encode, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more different polypeptides (all orpart). Any of the polypeptides, proteins, or peptides disclosed in Table2 may be produced recombinantly as part of the disclosed invention.Furthermore, any of the proteinaceous compounds in Table 2 may beencoded with one or more other polypeptides in Table 2 or disclosedherein on the same nucleic acid molecule such that a fusion protein iscreated. Recombinant proteins can be purified from expressing cells toyield active proteins.

Thus, embodiments of the invention include the use of nucleic acidsencoding all or part of SEQ ID NO:1. Such nucleic acids include all orpart of SEQ ID NO:2, which corresponds to the cDNA sequence encoding agelonin polypeptide (GenBank accession number L12243. Thus, it iscontemplated that any of the methods and compositions discussed hereinwith respect to nucleic acids may be applied with respect to SEQ IDNO:2.

As used herein, the term “DNA segment” refers to a DNA molecule that hasbeen isolated free of total genomic DNA of a particular species.Therefore, a DNA segment encoding a polypeptide refers to a DNA segmentthat contains wild-type, polymorphic, or mutant polypeptide-codingsequences yet is isolated away from, or purified free from, totalmammalian or human genomic DNA. Included within the term “DNA segment”are a polypeptide or polypeptides, DNA segments smaller than apolypeptide, and recombinant vectors, including, for example, plasmids,cosmids, phage, viruses, and the like.

As used in this application, the term “polynucleotide” refers to anucleic acid molecule that has been isolated free of total genomicnucleic acid. Therefore, a “polynucleotide encoding a nativepolypeptide” refers to a DNA segment that contains wild-type orpolymorphic polypeptide-coding sequences isolated away from, or purifiedfree from, total mammalian or human genomic DNA. Therefore, for example,when the present application refers to the function or activity ofgelonin, “native gelonin polypeptide,” or “modified gelonin polypeptide”that is encoded by a gelonin polynucleotide, it is meant that thepolynucleotide encodes a molecule that has enzymatic activity as a RIP.

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression or where non-coding regions such asintrons are to be targeted in an antisense strategy.

It also is contemplated that a particular polypeptide from a givenspecies may be represented by natural variants that have slightlydifferent nucleic acid sequences but, nonetheless, encode the sameprotein (see Table 1 above).

Similarly, a polynucleotide comprising an isolated or purifiedwild-type, polymorphic, or mutant polypeptide gene refers to a DNAsegment including wild-type, polymorphic, or mutant polypeptide codingsequences and, in certain aspects, regulatory sequences, isolatedsubstantially away from other naturally occurring genes or proteinencoding sequences. In this respect, the term “gene” is used forsimplicity to refer to a functional protein, polypeptide, orpeptide-encoding unit. As will be understood by those in the art, thisfunctional term includes genomic sequences, CDNA sequences, and smallerengineered gene segments that express, or may be adapted to express,proteins, polypeptides, domains, peptides, fusion proteins, and mutants.A nucleic acid encoding all or part of a native or modified polypeptidemay contain a contiguous nucleic acid sequence encoding all or a portionof such a polypeptide of the following lengths: about, at least, or atmost 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560,570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700,710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840,850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980,990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1095,1100, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500,7000, 7500, 8000, 9000, 10000, or more nucleotides, nucleosides, or basepairs. Such lengths of contiguous residues may apply to any nucleic acidsequence described or discussed herein, including SEQ ID NOS:2-10.

In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences that encode awild-type, polymorphic, or modified polypeptide or peptide that includeswithin its amino acid sequence a contiguous amino acid sequence inaccordance with, or essentially corresponding to a native polypeptide.Thus, an isolated DNA segment or vector containing a DNA segment mayencode, for example, a modified gelonin polypeptide that has theribosome-inactivating activity and specificity of a native geloninpolypeptide, yet have differing amino acids. The term “recombinant” maybe used in conjunction with a polypeptide or the name of a specificpolypeptide, and this generally refers to a polypeptide produced from anucleic acid molecule that has been manipulated in vitro or that is thereplicated product of such a molecule.

In other embodiments, the invention concerns isolated DNA segments andrecombinant vectors incorporating DNA sequences that encode apolypeptide or peptide that includes within its amino acid sequence acontiguous amino acid sequence in accordance with, or essentiallycorresponding to the polypeptide.

The nucleic acid segments used in the present invention, regardless ofthe length of the coding sequence itself, may be combined with othernucleic acid sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol.

It is contemplated that the nucleic acid constructs of the presentinvention may encode full-length polypeptide from any source or encode atruncated version of the polypeptide, for example a truncated geloninpolypeptide, such that the transcript of the coding region representsthe truncated version. The truncated transcript may then be translatedinto a truncated protein. Alternatively, a nucleic acid sequence mayencode a full-length polypeptide sequence with additional heterologouscoding sequences, for example to allow for purification of thepolypeptide, transport, secretion, post-translational modification, orfor therapeutic benefits such as targetting or efficacy. As discussedabove, a tag or other heterologous polypeptide may be added to themodified polypeptide-encoding sequence, wherein “heterologous” refers toa polypeptide that is not the same as the modified polypeptide.

In a non-limiting example, one or more nucleic acid constructs may beprepared that include a contiguous stretch of nucleotides identical toor complementary to the a particular gene, such as the toxin gelonin. Anucleic acid construct may be at least 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400,500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000,7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 30,000, 50,000, 100,000,250,000, 500,000, 750,000, to at least 1,000,000 nucleotides in length,as well as constructs of greater size, up to and including chromosomalsizes (including all intermediate lengths and intermediate ranges),given the advent of nucleic acids constructs such as a yeast artificialchromosome are known to those of ordinary skill in the art. It will bereadily understood that “intermediate lengths” and “intermediateranges,” as used herein, means any length or range including or betweenthe quoted values (i.e., all integers including and between suchvalues).

The DNA segments used in the present invention encompass biologicallyfunctional equivalent modified polypeptides and peptides, for example, amodified gelonin toxin. Such sequences may arise as a consequence ofcodon redundancy and functional equivalency that are known to occurnaturally within nucleic acid sequences and the proteins thus encoded.Alternatively, functionally equivalent proteins or peptides may becreated via the application of recombinant DNA technology, in whichchanges in the protein structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by human may be introduced through the application ofsite-directed mutagenesis techniques, e.g., to introduce improvements tothe antigenicity of the protein, to reduce toxicity effects of theprotein in vivo to a subject given the protein, or to increase theefficacy of any treatment involving the protein.

1. Vectors

Native and modified polypeptides may be encoded by a nucleic acidmolecule comprised in a vector. The term “vector” is used to refer to acarrier nucleic acid molecule into which a nucleic acid sequence can beinserted for introduction into a cell where it can be replicated. Anucleic acid sequence can be “exogenous,” which means that it is foreignto the cell into which the vector is being introduced or that thesequence is homologous to a sequence in the cell but in a positionwithin the host cell nucleic acid in which the sequence is ordinarilynot found. Vectors include plasmids, cosmids, viruses (bacteriophage,animal viruses, and plant viruses), and artificial chromosomes (e.g.,YACs). One of skill in the art would be well equipped to construct avector through standard recombinant techniques, which are described inSambrook et al., 1989 and Ausubel et al., 1996, both incorporated hereinby reference. In addition to encoding a modified polypeptide such asmodified gelonin, a vector may encode non-modified polypeptide sequencessuch as a tag or targetting molecule. Useful vectors encoding suchfusion proteins include pIN vectors (Inouye et al., 1985), vectorsencoding a stretch of histidines, and pGEX vectors, for use ingenerating glutathione S-transferase (GST) soluble fusion proteins forlater purification and separation or cleavage. A targetting molecule isone that directs the modified polypeptide to a particular organ, tissue,cell, or other location in a subject's body.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host organism. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

a. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind such as RNA polymerase and other transcriptionfactors. The phrases “operatively positioned,” “operatively linked,”“under control,” and “under transcriptional control” mean that apromoter is in a correct functional location and/or orientation inrelation to a nucleic acid sequence to control transcriptionalinitiation and/or expression of that sequence. A promoter may or may notbe used in conjunction with an “enhancer,” which refers to a cis-actingregulatory sequence involved in the transcriptional activation of anucleic acid sequence.

A promoter may be one naturally associated with a gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other prokaryotic, viral, or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. In addition to producing nucleicacid sequences of promoters and enhancers synthetically, sequences maybe produced using recombinant cloning and/or nucleic acid amplificationtechnology, including PCR™, in connection with the compositionsdisclosed herein (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906,each incorporated herein by reference). Furthermore, it is contemplatedthe control sequences that direct transcription and/or expression ofsequences within non-nuclear organelles such as mitochondria,chloroplasts, and the like, can be employed as well.

Naturally, it may be important to employ a promoter and/or enhancer thateffectively directs the expression of the DNA segment in the cell type,organelle, and organism chosen for expression. Those of skill in the artof molecular biology generally know the use of promoters, enhancers, andcell type combinations for protein expression, for example, see Sambrooket al. (1989), incorporated herein by reference. The promoters employedmay be constitutive, tissue-specific, inducible, and/or useful under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins and/or peptides. The promoter may be heterologousor endogenous.

Tables 5 lists several elements/promoters that may be employed, in thecontext of the present invention, to regulate the expression of a gene.This list is not intended to be exhaustive of all the possible elementsinvolved in the promotion of expression but, merely, to be exemplarythereof. Table 6 provides examples of inducible elements, which areregions of a nucleic acid sequence that can be activated in response toa specific stimulus.

TABLE 5 Promoter and/or Enhancer Promoter/Enhancer ReferencesImmunoglobulin Heavy Chain Banerji et al., 1983; Gilles et al., 1983;Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler et al.,1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al.;1990 Immunoglobulin Light Chain Queen et al., 1983; Picard et al., 1984T-Cell Receptor Luria et al., 1987; Winoto et al., 1989; Redondo et al.;1990 HLA DQ a and/or DQ β Sullivan et al., 1987 β-Interferon Goodbournet al., 1986; Fujita et al., 1987; Goodbourn et al., 1988 Interleukin-2Greene et al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin etal., 1990 MHC Class II 5 Koch et al., 1989 MHC Class II HLA-DRa Shermanet al., 1989 β-Actin Kawamoto et al., 1988; Ng et al.; 1989 MuscleCreatine Kinase (MCK) Jaynes et al., 1988; Horlick et al., 1989; Johnsonet al., 1989 Prealbumin (Transthyretin) Costa et al., 1988 Elastase IOrnitz et al., 1987 Metallothionein (MTII) Karin et al., 1987; Culottaet al., 1989 Collagenase Pinkert et al., 1987; Angel et al., 1987Albumin Pinkert et al., 1987; Tronche et al., 1989, 1990 α-FetoproteinGodbout et al., 1988; Campere et al., 1989 γ-Globin Bodine et al., 1987;Perez-Stable et al., 1990 β-Globin Trudel et al., 1987 c-fos Cohen etal., 1987; Treisman, 1986; Deschamps et al., 1985 c-HA-ras Trimble andHozumi, 1987 Insulin Edlund et al., 1985 Neural Cell Adhesion MoleculeHirsh et al., 1990 (NCAM) α₁-Antitrypain Latimer et al., 1990 H2B (TH2B)Histone Hwang et al., 1990 Mouse and/or Type I Collagen Ripe et al.,1989 Glucose-Regulated Proteins Chang et al., 1989 (GRP94 and GRP78) RatGrowth Hormone Larsen et al., 1986 Human Serum Amyloid A (SAA) Edbrookeet al., 1989 Troponin I (TN I) Yutzey et al., 1989 Platelet-DerivedGrowth Factor Pech et al., 1989 (PDGF) Duchenne Muscular DystrophyKlamut et al., 1990 SV40 Banerji et al., 1981; Moreau et al., 1981;Sleigh et al., 1985; Firak et al., 1986; Herr et al., 1986; Imbra etal., 1986; Kadesch et al., 1986; Wang et al., 1986; Ondek et al., 1987;Kuhl et al., 1987; Schaffner et al., 1988 Polyoma Swartzendruber et al.,1975; Vasseur et al., 1980; Katinka et al., 1980, 1981; Tyndell et al.,1981; Dandolo et al., 1983; de Villiers et al., 1984; Hen et al., 1986;Satake et al., 1988; Campbell and/or Villarreal, 1988 RetrovirusesKriegler et al., 1982, 1983; Levinson et al., 1982; Kriegler et al.,1983, 1984a, b, 1988; Bosze et al., 1986; Miksicek et al., 1986;Celander et al., 1987; Thiesen et al., 1988; Celander et al., 1988; Choiet al., 1988; Reisman et al., 1989 Papilloma Virus Campo et al., 1983;Lusky et al., 1983; Spandidos and/or Wilkie, 1983; Spalholz et al.,1985; Lusky et al., 1986; Cripe et al., 1987; Gloss et al., 1987;Hirochika et al., 1987; Stephens et al., 1987 Hepatitis B Virus Bulla etal., 1986; Jameel et al., 1986; Shaul et al., 1987; Spandau et al.,1988; Vannice et al., 1988 Human Immunodeficiency Virus Muesing et al.,1987; Hauber et al., 1988; Jakobovits et al., 1988; Feng et al., 1988;Takebe et al., 1988; Rosen et al., 1988; Berkhout et al., 1989; Laspiaet al., 1989; Sharp et al., 1989; Braddock et al., 1989 Cytomegalovirus(CMV) Weber et al., 1984; Boshart et al., 1985; Foecking et al., 1986Gibbon Ape Leukemia Virus Holbrook et al., 1987; Quinn et al., 1989

TABLE 6 Inducible Elements Element Inducer References MT II PhorbolEster (TPA) Palmiter et al., 1982; Haslinger Heavy metals et al., 1985;Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin etal., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouse mammaryGlucocorticoids Huang et al., 1981; Lee et al., tumor virus) 1981;Majors et al., 1983; Chandler et al., 1983; Lee et al., 1984; Ponta etal., 1985; Sakai et al., 1988 β-Interferon poly(rI)x Tavernier et al.,1983 poly(rc) Adenovirus 5 E2 ElA Imperiale et al., 1984 CollagenasePhorbol Ester (TPA) Angel et al., 1987a Stromelysin Phorbol Ester (TPA)Angel et al., 1987b SV40 Phorbol Ester (TPA) Angel et al., 1987b MurineMX Gene Interferon, Newcastle Hug et al., 1988 Disease Virus GRP78 GeneA23187 Resendez et al., 1988 α-2-Macroglobulin IL-6 Kunz et al., 1989Vimentin Serum Rittling et al., 1989 MHC Class I Gene H-2κb InterferonBlanar et al., 1989 HSP70 ElA, SV40 Large T Taylor et al., 1989, 1990a,1990b Antigen Proliferin Phorbol Ester-TPA Mordacq et al., 1989 TumorNecrosis Factor PMA Hensel et al., 1989 Thyroid Stimulating ThyroidHormone Chatterjee et al., 1989 Hormone α Gene

The identity of tissue-specific promoters or elements, as well as assaysto characterize their activity, is well known to those of skill in theart. Examples of such regions include the human LIMK2 gene (Nomoto etal. 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), murineepididymal retinoic acid-binding gene (Lareyre et al., 1999), human CD4(Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen (Tsumaki, et al.,1998), D1A dopamine receptor gene (Lee, et al., 1997), insulin-likegrowth factor II (Wu et al., 1997), human platelet endothelial celladhesion molecule-1 (Almendro et al., 1996).

b. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, herein incorporated by reference).

c. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector. (See Carbonelli et al., 1999, Levenson et al., 1998,and Cocea, 1997, incorporated herein by reference.) “Restriction enzymedigestion” refers to catalytic cleavage of a nucleic acid molecule withan enzyme that functions only at specific locations in a nucleic acidmolecule. Many of these restriction enzymes are commercially available.Use of such enzymes is widely understood by those of skill in the art.Frequently, a vector is linearized or fragmented using a restrictionenzyme that cuts within the MCS to enable exogenous sequences to beligated to the vector. “Ligation” refers to the process of formingphosphodiester bonds between two nucleic acid fragments, which may ormay not be contiguous with each other. Techniques involving restrictionenzymes and ligation reactions are well known to those of skill in theart of recombinant technology.

d. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression. (SeeChandler et al., 1997, herein incorporated by reference.)

e. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and/or to minimize read through from thecassette into other sequences.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

f. Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and/or any suchsequence may be employed. Preferred embodiments include the SV40polyadenylation signal and/or the bovine growth hormone polyadenylationsignal, convenient and/or known to function well in various targetcells. Polyadenylation may increase the stability of the transcript ormay facilitate cytoplasmic transport.

g. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

h. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transform ants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

2. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organisms that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid, such as a modified protein-encoding sequence, istransferred or introduced into the host cell. A transformed cellincludes the primary subject cell and its progeny.

Host cells may be derived from prokaryotes or eukaryotes, includingyeast cells, insect cells, and mammalian cells, depending upon whetherthe desired result is replication of the vector or expression of part orall of the vector-encoded nucleic acid sequences. Numerous cell linesand cultures are available for use as a host cell, and they can beobtained through the American Type Culture Collection (ATCC), which isan organization that serves as an archive for living cultures andgenetic materials (www.atcc.org). An appropriate host can be determinedby one of skill in the art based on the vector backbone and the desiredresult. A plasmid or cosmid, for example, can be introduced into aprokaryote host cell for replication of many vectors. Bacterial cellsused as host cells for vector replication and/or expression includeDH5α, JM109, and KC8, as well as a number of commercially availablebacterial hosts such as SURE® Competent Cells and SOLOPACK™ Gold Cells(STRATAGENE®, La Jolla). Alternatively, bacterial cells such as E. coliLE392 could be used as host cells for phage viruses. Appropriate yeastcells include Saccharomyces cerevisiae, Saccharomyces pombe, and Pichiapastoris.

Examples of eukaryotic host cells for replication and/or expression of avector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Manyhost cells from various cell types and organisms are available and wouldbe known to one of skill in the art. Similarly, a viral vector may beused in conjunction with either a eukaryotic or prokaryotic host cell,particularly one that is permissive for replication or expression of thevector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

3. Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with the present invention to producenucleic acid sequences, or their cognate polypeptides, proteins andpeptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986, 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MAXBAC®2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROMCLONTECH®.

In addition to the disclosed expression systems of the invention, otherexamples of expression systems include STRATAGENE®'s COMPLETE CONTROL™Inducible Mammalian Expression System, which involves a syntheticecdysone-inducible receptor, or its pET Expression System, an E. coliexpression system. Another example of an inducible expression system isavailable from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

4. Viral Vectors

There are a number of ways in which expression vectors may be introducedinto cells. In certain embodiments of the invention, the expressionvector comprises a virus or engineered vector derived from a viralgenome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden,1986; Temin, 1986). The first viruses used as gene vectors were DNAviruses including the papovaviruses (simian virus 40, bovine papillomavirus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) andadenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have arelatively low capacity for foreign DNA sequences and have a restrictedhost spectrum. Furthermore, their oncogenic potential and cytopathiceffects in permissive cells raise safety concerns. They can accommodateonly up to 8 kb of foreign genetic material but can be readilyintroduced in a variety of cell lines and laboratory animals (Nicolasand Rubenstein, 1988; Temin, 1986).

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells; they can also be used as vectors. Other viral vectorsmay be employed as expression constructs in the present invention.Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988;Baichwal and Sugden, 1986; Coupar et al., 1988) adeno-associated virus(AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska,1984) and herpesviruses may be employed. They offer several attractivefeatures for various mammalian cells (Friedmann, 1989; Ridgeway, 1988;Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

B. Nucleic Acid Detection

In addition to their use in directing the expression of designer toxinand modified proteins, polypeptides and/or peptides, the nucleic acidsequences disclosed herein have a variety of other uses. For example,they have utility as probes or primers for embodiments involving nucleicacid hybridization. Detection of nucleic acids encoding modifiedproteins or designer toxins are encompassed by the invention.

1. Hybridization

The use of a probe or primer of between 13 and 100 nucleotides,preferably between 17 and 100 nucleotides in length, or in some aspectsof the invention up to 1-2 kilobases or more in length, allows theformation of a duplex molecule that is both stable and selective.Molecules having complementary sequences over contiguous stretchesgreater than 20 bases in length are generally preferred, to increasestability and/or selectivity of the hybrid molecules obtained. One willgenerally prefer to design nucleic acid molecules for hybridizationhaving one or more complementary sequences of 20 to 30 nucleotides, oreven longer where desired. Such fragments may be readily prepared, forexample, by directly synthesizing the fragment by chemical means or byintroducing selected sequences into recombinant vectors for recombinantproduction.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNAs and/or RNAs or to provide primers for amplification ofDNA or RNA from samples. Depending on the application envisioned, onewould desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of the probe or primers for the targetsequence.

For applications requiring high selectivity, one will typically desireto employ relatively high stringency conditions to form the hybrids. Forexample, relatively low salt and/or high temperature conditions, such asprovided by about 0.02 M to about 0.10 M NaCl at temperatures of about50° C. to about 70° C. Such high stringency conditions tolerate little,if any, mismatch between the probe or primers and the template or targetstrand and would be particularly suitable for isolating specific genesor for detecting specific mRNA transcripts. It is generally appreciatedthat conditions can be rendered more stringent by the addition ofincreasing amounts of formamide.

For certain applications, for example, site-directed mutagenesis, it isappreciated that lower stringency conditions are preferred. Under theseconditions, hybridization may occur even though the sequences of thehybridizing strands are not perfectly complementary, but are mismatchedat one or more positions. Conditions may be rendered less stringent byincreasing salt concentration and/or decreasing temperature. Forexample, a medium stringency condition could be provided by about 0.1 to0.25 M NaCl at temperatures of about 37° C. to about 55° C., while a lowstringency condition could be provided by about 0.15 M to about 0.9 Msalt, at temperatures ranging from about 20° C. to about 55° C.Hybridization conditions can be readily manipulated depending on thedesired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 1.0 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C.

In certain embodiments, it will be advantageous to employ nucleic acidsof defined sequences of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of being detected. In preferredembodiments, one may desire to employ a fluorescent label or an enzymetag such as urease, alkaline phosphatase or peroxidase, instead ofradioactive or other environmentally undesirable reagents. In the caseof enzyme tags, colorimetric indicator substrates are known that can beemployed to provide a detection means that is visibly orspectrophotometrically detectable, to identify specific hybridizationwith complementary nucleic acid containing samples.

In general, it is envisioned that the probes or primers described hereinwill be useful as reagents in solution hybridization, as in PCR™, fordetection of expression of corresponding genes, as well as inembodiments employing a solid phase. In embodiments involving a solidphase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to hybridization with selected probes under desiredconditions. The conditions selected will depend on the particularcircumstances (depending, for example, on the G+C content, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, etc.). Optimization of hybridization conditions for theparticular application of interest is well known to those of skill inthe art. After washing of the hybridized molecules to removenon-specifically bound probe molecules, hybridization is detected,and/or quantified, by determining the amount of bound label.Representative solid phase hybridization methods are disclosed in U.S.Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods ofhybridization that may be used in the practice of the present inventionare disclosed in U.S. Pat. Nos. 5,849,481, 5,849,486 and 5,851,772. Therelevant portions of these and other references identified in thissection of the Specification are incorporated herein by reference.

2. Amplification of Nucleic Acids

Nucleic acids used as a template for amplification may be isolated fromcells, tissues or other samples according to standard methodologies(Sambrook et al., 1989). In certain embodiments, analysis is performedon whole cell or tissue homogenates or biological fluid samples withoutsubstantial purification of the template nucleic acid. The nucleic acidmay be genomic DNA or fractionated or whole cell RNA. Where RNA is used,it may be desired to first convert the RNA to a complementary DNA.

The term “primer,” as used herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty and/or thirty base pairs in length, but longersequences can be employed. Primers may be provided in double-strandedand/or single-stranded form, although the single-stranded form ispreferred.

Pairs of primers designed to selectively hybridize to nucleic acidscorresponding to SEQ ID NO:1 or any other SEQ ID NO are contacted withthe template nucleic acid under conditions that permit selectivehybridization. Depending upon the desired application, high stringencyhybridization conditions may be selected that will only allowhybridization to sequences that are completely complementary to theprimers. In other embodiments, hybridization may occur under reducedstringency to allow for amplification of nucleic acids contain one ormore mismatches with the primer sequences. Once hybridized, thetemplate-primer complex is contacted with one or more enzymes thatfacilitate template-dependent nucleic acid synthesis. Multiple rounds ofamplification, also referred to as “cycles,” are conducted until asufficient amount of amplification product is produced.

The amplification product may be detected or quantified. In certainapplications, the detection may be performed by visual means.Alternatively, the detection may involve indirect identification of theproduct via chemiluminescence, radioactive scintigraphy of incorporatedradiolabel or fluorescent label or even via a system using electricaland/or thermal impulse signals (Affymax technology; Bellus, 1994).

A number of template dependent processes are available to amplify theoligonucleotide sequences present in a given template sample. One of thebest known amplification methods is the polymerase chain reaction(referred to as PCR™) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1988, each ofwhich is incorporated herein by reference in their entirety.

A reverse transcriptase PCR™ amplification procedure may be performed toquantify the amount of mRNA amplified. Methods of reverse transcribingRNA into cDNA are well known (see Sambrook et al., 1989). Alternativemethods for reverse transcription utilize thermostable DNA polymerases.These methods are described in WO 90/07641. Polymerase chain reactionmethodologies are well known in the art. Representative methods ofRT-PCR are described in U.S. Pat. No. 5,882,864.

Another method for amplification is ligase chain reaction (“LCR”),disclosed in European Application No. 320 308, incorporated herein byreference in its entirety. U.S. Pat. No. 4,883,750 describes a methodsimilar to LCR for binding probe pairs to a target sequence. A methodbased on PCR™ and oligonucleotide ligase assay (OLA), disclosed in U.S.Pat. No. 5,912,148, may also be used.

Alternative methods for amplification of target nucleic acid sequencesthat may be used in the practice of the present invention are disclosedin U.S. Pat. Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497,5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905,5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, GB ApplicationNo. 2 202 328, and in PCT Application No. PCT/US89/01025, each of whichis incorporated herein by reference in its entirety.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, mayalso be used as an amplification method in the present invention. Inthis method, a replicative sequence of RNA that has a regioncomplementary to that of a target is added to a sample in the presenceof an RNA polymerase. The polymerase will copy the replicative sequencewhich may then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention (Walker et al., 1992). StrandDisplacement Amplification (SDA), disclosed in U.S. Pat. No. 5,916,779,is another method of carrying out isothermal amplification of nucleicacids which involves multiple rounds of strand displacement andsynthesis, i.e., nick translation.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989; PCT Application WO88/10315, incorporated herein by reference in their entirety). EuropeanApplication No. 329 822 disclose a nucleic acid amplification processinvolving cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA,and double-stranded DNA (dsDNA), which may be used in accordance withthe present invention.

PCT Application WO 89/06700 (incorporated herein by reference in itsentirety) disclose a nucleic acid sequence amplification scheme based onthe hybridization of a promoter region/primer sequence to a targetsingle-stranded DNA (“ssDNA”) followed by transcription of many RNAcopies of the sequence. This scheme is not cyclic, i.e., new templatesare not produced from the resultant RNA transcripts. Other amplificationmethods include “RACE” and “one-sided PCR” (Frohman, 1990; Ohara et al.,1989).

3. Detection of Nucleic Acids

Following any amplification, it may be desirable to separate theamplification product from the template and/or the excess primer. In oneembodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (Sambrook et al., 1989). Separated amplification products may becut out and eluted from the gel for further manipulation. Using lowmelting point agarose gels, the separated band may be removed by heatingthe gel, followed by extraction of the nucleic acid.

Separation of nucleic acids may also be effected by chromatographictechniques known in art. There are many kinds of chromatography whichmay be used in the practice of the present invention, includingadsorption, partition, ion-exchange, hydroxylapatite, molecular sieve,reverse-phase, column, paper, thin-layer, and gas chromatography as wellas HPLC.

In certain embodiments, the amplification products are visualized. Atypical visualization method involves staining of a gel with ethidiumbromide and visualization of bands under UV light. Alternatively, if theamplification products are integrally labeled with radio- orfluorometrically-labeled nucleotides, the separated amplificationproducts can be exposed to x-ray film or visualized under theappropriate excitatory spectra.

In one embodiment, following separation of amplification products, alabeled nucleic acid probe is brought into contact with the amplifiedmarker sequence. The probe preferably is conjugated to a chromophore butmay be radiolabeled. In another embodiment, the probe is conjugated to abinding partner, such as an antibody or biotin, or another bindingpartner carrying a detectable moiety.

In particular embodiments, detection is by Southern blotting andhybridization with a labeled probe. The techniques involved in Southernblotting are well known to those of skill in the art (see Sambrook etal., 1989). One example of the foregoing is described in U.S. Pat. No.5,279,721, incorporated by reference herein, which discloses anapparatus and method for the automated electrophoresis and transfer ofnucleic acids. The apparatus permits electrophoresis and blottingwithout external manipulation of the gel and is ideally suited tocarrying out methods according to the present invention.

Other methods of nucleic acid detection that may be used in the practiceof the instant invention are disclosed in U.S. Pat. Nos. 5,840,873,5,843,640, 5,843,651, 5,846,708, 5,846,717, 5,846,726, 5,846,729,5,849,487, 5,853,990, 5,853,992, 5,853,993, 5,856,092, 5,861,244,5,863,732, 5,863,753, 5,866,331, 5,905,024, 5,910,407, 5,912,124,5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869, 5,929,227,5,932,413 and 5,935,791, each of which is incorporated herein byreference.

4. Other Assays

Other methods for genetic screening may be used within the scope of thepresent invention, for example, to detect mutations in genomic DNA, cDNAand/or RNA samples. Methods used to detect point mutations includedenaturing gradient gel electrophoresis (“DGGE”), restriction fragmentlength polymorphism analysis (“RFLP”), chemical or enzymatic cleavagemethods, direct sequencing of target regions amplified by PCR™ (seeabove), single-strand conformation polymorphism analysis (“SSCP”) andother methods well known in the art.

One method of screening for point mutations is based on RNase cleavageof base pair mismatches in RNA/DNA or RNA/RNA heteroduplexes. As usedherein, the term “mismatch” is defined as a region of one or moreunpaired or mispaired nucleotides in a double-stranded RNA/RNA, RNA/DNAor DNA/DNA molecule. This definition thus includes mismatches due toinsertion/deletion mutations, as well as single or multiple base pointmutations.

U.S. Pat. No. 4,946,773 describes an RNase A mismatch cleavage assaythat involves annealing single-stranded DNA or RNA test samples to anRNA probe, and subsequent treatment of the nucleic acid duplexes withRNase A. For the detection of mismatches, the single-stranded productsof the RNase A treatment, electrophoretically separated according tosize, are compared to similarly treated control duplexes. Samplescontaining smaller fragments (cleavage products) not seen in the controlduplex are scored as positive.

Other investigators have described the use of RNase I in mismatchassays. The use of RNase I for mismatch detection is described inliterature from Promega Biotech. Promega markets a kit containing RNaseI that is reported to cleave three out of four known mismatches. Othershave described using the MutS protein or other DNA-repair enzymes fordetection of single-base mismatches.

Alternative methods for detection of deletion, insertion or substitutionmutations that may be used in the practice of the present invention aredisclosed in U.S. Pat. Nos. 5,849,483, 5,851,770, 5,866,337, 5,925,525and 5,928,870, each of which is incorporated herein by reference in itsentirety.

C. Methods of Gene Transfer

Suitable methods for nucleic acid delivery to effect expression ofcompositions of the present invention are believed to include virtuallyany method by which a nucleic acid (e.g., DNA, including viral andnonviral vectors) can be introduced into an organelle, a cell, a tissueor an organism, as described herein or as would be known to one ofordinary skill in the art. Such methods include, but are not limited to,direct delivery of DNA such as by injection (U.S. Pat. Nos. 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610,5,589,466 and 5,580,859, each incorporated herein by reference),including microinjection (Harlan and Weintraub, 1985; U.S. Pat. No.5,789,215, incorporated herein by reference); by electroporation (U.S.Pat. No. 5,384,253, incorporated herein by reference); by calciumphosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama,1987; Rippe et al., 1990); by using DEAE-dextran followed bypolyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimeret al., 1987); by liposome mediated transfection (Nicolau and Sene,1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980;Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment(PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat. Nos.5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880, andeach incorporated herein by reference); by agitation with siliconcarbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and5,464,765, each incorporated herein by reference); byAgrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and5,563,055, each incorporated herein by reference); or by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos.4,684,611 and 4,952,500, each incorporated herein by reference); bydesiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985).Through the application of techniques such as these, organelle(s),cell(s), tissue(s) or organism(s) may be stably or transientlytransformed.

III. Ribosome-Inactivating Proteins

Ribosome-inhibitory toxins (RITs) are potent inhibitors of proteinsynthesis in eukaryotes. The enzymatic domain of these proteins acts asa cytotoxic n-glycosidase that is able to inactivate catalyticallyribosomes once they gain entry to the intracellular compartment. This isaccomplished by cleaving the n-glycosidic bond of the adenine atposition 4324 in the 28srRNA, which irreversibly inactivates theribosome apparently by disrupting the binding site for elongationfactors. RITs, which have been isolated from bacteria, are prevalent inhigher plants. In plants, there are two types: Type I toxins possess asingle polypeptide chain that has ribosome inhibiting activity, and TypeII toxins have an A chain, comparable to the Type I protein, that islinked by a disulfide bond to a B chain possessing cell-bindingproperties. Examples of Type I RITs are gelonin, dodecandrin,tricosanthin, tricokirin, bryodin, mirabilis antiviral protein, barleyribosome-inactivating protein (BRIP), pokeweed antiviral proteins(PAPs), saporins, luffins, and momordins. Type II toxins include ricinand abrin. Toxins may be conjugated or expressed as a fusion proteinwith any of the polypeptides discussed herein. Alternatively, themodified toxins of the present invention may be conjugated to a smallmolecule, such as a chemotherapeutic or a targeting agent.

A. Immunotoxins

The toxins of the invention are particularly suited for use ascomponents of cytotoxic therapeutic agents. These cytotoxic agents maybe used in vivo to selectively eliminate a particular cell type to whichthe toxin component is targeted by the specific binding capacity of asecond component. To form cytotoxic agents, modified toxins of thepresent invention may be conjugated to monoclonal antibodies, includingchimeric and CDR-grafted antibodies, and antibody domains/fragments(e.g., Fab, Fab′, F(ab′).sub.2, single chain antibodies, and Fv orsingle variable domains). Immunoconjugates including toxins may bedescribed as immunotoxins. An immunotoxin may also consist of a fusionprotein rather than an immunoconjugate.

Modified toxins conjugated to monoclonal antibodies geneticallyengineered to include free cysteine residues are also within the scopeof the present invention. Examples of Fab′ and F(ab′).sub.2 fragmentsuseful in the present invention are described in WO 89/00999, which isincorporated by reference herein.

Alternatively, the modified toxins may be conjugated or fused tohumanized or human engineered antibodies. Such humanized antibodies maybe constructed from mouse antibody variable domains.

1. Antibody Regions

Regions from the various members of the immunoglobulin family areencompassed by the present invention. Both variable regions fromspecific antibodies are covered within the present invention, includingcomplementarity determining regions (CDRs), as are antibody neutralizingregions, including those that bind effector molecules such as Fcregions. Antigen specific-encoding regions from antibodies, such asvariable regions from IgGs, IgMs, or IgAs, can be employed with anothermolecule such as a toxin in combination with an antibody neutralizationregion or with one of the therapeutic compounds described above.

In yet another embodiment, one gene may comprise a single-chainantibody. Methods for the production of single-chain antibodies are wellknown to those of skill in the art. The skilled artisan is referred toU.S. Pat. No. 5,359,046, (incorporated herein by reference) for suchmethods. A single chain antibody is created by fusing together thevariable domains of the heavy and light chains using a short peptidelinker, thereby reconstituting an antigen binding site on a singlemolecule.

Single-chain antibody variable fragments (scFvs) in which the C-terminusof one variable domain is tethered to the N-terminus of the other via a15 to 25 amino acid peptide or linker, have been developed withoutsignificantly disrupting antigen binding or specificity of the binding(Bedzyk et al., 1990; Chaudhary et al., 1990). These Fvs lack theconstant regions (Fc) present in the heavy and light chains of thenative antibody. Immunotoxins employing single-chain antibodies aredescribed in U.S. Pat. No. 6,099,842, specifically incorporated byreference.

Antibodies to a wide variety of molecules are contemplated, such asoncogenes, tumor-associated antigens, cytokines, growth factors,hormones, enzymes, transcription factors or receptors. Also contemplatedare secreted antibodies targeted against serum, angiogenic factors(VEGF/VPF; βFGF; αFGF; and others), coagulation factors, and endothelialantigens necessary for angiogenesis (i.e., V3 integrin). Specificallycontemplated are growth factors such as transforming growth factor,fibroblast growth factor, and platelet derived growth factor (PDGF) andPDGF family members.

The present invention further embodies composition targeting specificpathogens through the use of antigen-specific sequences or targetingspecific cell types, such as those expressing cell surface markers toidentify the cell. Examples of such cell surface markers would includetumor-associated antigens or cell-type specific markers such as CD4 orCD8.

The antibodies employed in the present invention as part of animmunotoxin may be targeted to any antigen. The antigen may be specificto an organism, to a cell type, to a disease or condition, or to apathogen. Exemplary antigens include cell surface cellular proteins, forexample tumor-associated antigens, viral proteins, microbial proteins,post-translational modifications or carbohydrates, and receptors. Commontumor markers include carcinoembryonic antigen, prostate specificantigen, urinary tumor associated antigen, fetal antigen, tyrosinase(p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP,estrogen receptor, laminin receptor, erb B and p155. Other antigens thatmay be targeted include the receptors for EGF and VEGF, TIE-1 and -2,CD-33, CD38, CD-20, CD-52, GP-240, Lym-1, MMO-2, and MMP-9.

B. Other Targetting Moieties

The use of a region of a protein that mediates protein-proteininteractions, including ligand-receptor interactions, also iscontemplated by the present invention. This region could be used as aninhibitor or competitor of a protein-protein interaction or as aspecific targeting motif. Consequently, the invention covers using thetargetting moiety to recruit the toxin or other therapeutic ordiagnostic polypeptide to a particular body part, organ, tissue, orcell. Once the compositions of the present invention reach theparticular area through the targeting motif, the toxin or otherpolypeptide can function.

Targetting moieties may take advantage of protein-protein interactions.These include interactions betveen and among proteins such as receptorsand ligands; receptors and receptors; polymeric complexes; transcriptionfactors; kinases and downstream targets; enzymes and substrates; etc.For example, a ligand binding domain mediates the protein:proteininteraction between a ligand and its cognate receptor. Consequently,this domain could be used either to inhibit or compete with endogenousligand binding or to target more specifically cell types that express areceptor that recognizes the ligand binding domain operatively attachedto a therapeutic polypeptide, such as the gelonin toxin.

Examples of ligand binding domains include ligands such as VEGF/VPF;βFGF; αFGF; coagulation factors, and endothelial antigens necessary forangiogenesis (i.e., V3 integrin); growth factors such as transforminggrowth factor, fibroblast growth factor, colony stimulating factor, Kitligand (KL), flk-2/flt-3, and platelet derived growth factor (PDGF) andPDGF family members; ligands that bind to cell surface receptors such asMHC molecules, among other.

The most extensively characterized ligands are asialoorosomucoid (ASOR)(Wu and Wu, 1987) and transferrin (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, Nicolau et al. (1987) employed lactosyl-ceramide,a galactose-terminal asialganglioside, incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes.Also, the human prostate-specific antigen (Watt et al., 1986) may beused as the receptor for mediated delivery to prostate tissue.

In still further embodiments, a lectin molecule may be used to target acompound to a cell expressing a particular carbohydrate on its surface.

1. Cytokines

Another class of compounds that is contemplated to be operatively linkedto a therapeutic polypeptide, such as a toxin, includes interleukins andcytokines, such as interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,β-interferon, α-interferon, γ-interferon, angiostatin, thrombospondin,endostatin, METH-1, METH-2, Flk2/Flt3 ligand, GM-CSF, G-CSF, M-CSF, andtumor necrosis factor (TNF).

2. Growth Factors

In other embodiments of the present invention, growth factors or ligandscan be complexed with the therapeutic agent. Examples include VEGF/VPF,FGF, TGFβ, ligands that bind to a TIE, tumor-associated fibronectinisoforms, scatter factor, hepatocyte growth factor, fibroblast growthfactor, platelet factor (PF4), PDGF, KIT ligand (KL), colony stimulatingfactors (CSFs), LIF, and TIMP.

3. Inducers of Cellular Proliferation

Another group of proteins that may be used in conjunction with modifiedproteins of the present invention, such as modified gelonin toxin,comprises proteins that induce cellular proliferation. In someembodiments, the toxin is operatively linked to a ribozyme that caninactivate an inducer of cellular proliferation, while in others, thetoxin is linked to the inducer itself. Alternatively, a toxin may beattached to an antibody that recognizes an inducer of cellproliferation.

The commonality of all of these proteins is their ability to regulatecellular proliferation. For example, a form of PDGF, the sis oncogene,is a secreted growth factor. Oncogenes rarely arise from genes encodinggrowth factors, and at the present, sis is the only knownnaturally-occurring oncogenic growth factor. In one embodiment of thepresent invention, it is contemplated that anti-sense mRNA directed to aparticular inducer of cellular proliferation is used to preventexpression of the inducer of cellular proliferation.

The proteins FMS, ErbA, ErbB and neu are growth factor receptors.Mutations to these receptors result in loss of regulatable function. Forexample, a point mutation affecting the transmembrane domain of the Neureceptor protein results in the neu oncogene. The erbA oncogene isderived from the intracellular receptor for thyroid hormone. Themodified oncogenic ErbA receptor is believed to compete with theendogenous thyroid hormone receptor, causing uncontrolled growth.

The largest class of oncogenes includes the signal transducing proteins(e.g., Src, Abl and Ras). The protein Src is a cytoplasmicprotein-tyrosine kinase, and its transformation from proto-oncogene tooncogene in some cases, results via mutations at tyrosine residue 527.In contrast, transformation of GTPase protein ras from proto-oncogene tooncogene, in one example, results from a valine to glycine mutation atamino acid 12 in the sequence, reducing ras GTPase activity.

The proteins Jun, Fos and Myc are proteins that directly exert theireffects on nuclear functions as transcription factors.

4. Inhibitors of Cellular Proliferation

The tumor suppressors function to inhibit excessive cellularproliferation. The inactivation of these genes destroys their inhibitoryactivity, resulting in unregulated proliferation. It is contemplatedthat toxins may be attached to antibodies that recognize mutant tumorsuppressors or wild-type tumor suppressors. Alternatively, a toxin maybe linked to all or part of the tumor suppressor. The tumor suppressorsp53, p16 and C-CAM are described below.

High levels of mutant p53 have been found in many cells transformed bychemical carcinogenesis, ultraviolet radiation, and several viruses. Thep53 gene is a frequent target of mutational inactivation in a widevariety of human tumors and is already documented to be the mostfrequently mutated gene in common human cancers. It is mutated in over50% of human NSCLC (Hollstein et al., 1991) and in a wide spectrum ofother tumors.

The p53 gene encodes a 393-amino acid phosphoprotein that can formcomplexes with host proteins such as large-T antigen and E1B. Theprotein is found in normal tissues and cells, but at concentrationswhich are minute by comparison with transformed cells or tumor tissue

Wild-type p53 is recognized as an important growth regulator in manycell types. Missense mutations are common for the p53 gene and areessential for the transforming ability of the oncogene. A single geneticchange prompted by point mutations can create carcinogenic p53. Unlikeother oncogenes, however, p53 point mutations are known to occur in atleast 30 distinct codons, often creating dominant alleles that produceshifts in cell phenotype without a reduction to homozygosity.Additionally, many of these dominant negative alleles appear to betolerated in the organism and passed on in the germ line. Various mutantalleles appear to range from minimally dysfunctional to stronglypenetrant, dominant negative alleles (Weinberg, 1991).

Another inhibitor of cellular proliferation is p16. The majortransitions of the eukaryotic cell cycle are triggered bycyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4(CDK4), regulates progression through the G₁. The activity of thisenzyme may be to phosphorylate Rb at late G₁. The activity of CDK4 iscontrolled by an activating subunit, D-type cyclin, and by an inhibitorysubunit, the p16^(INK4) has been biochemically characterized as aprotein that specifically binds to and inhibits CDK4, and thus mayregulate Rb phosphorylation (Serrano et al., 1993; Serrano et al.,1995). Since the p16^(INK4) protein is a CDK4 inhibitor (Serrano, 1993),deletion of this gene may increase the activity of CDK4, resulting inhyperphosphorylation of the Rb protein. p16 also is known to regulatethe function of CDK6.

p16^(INK4) belongs to a newly described class of CDK-inhibitory proteinsthat also includes p16^(B), p19, p21^(WAF1), and p27^(KIP1). Thep16^(INK4) gene maps to 9p21, a chromosome region frequently deleted inmany tumor types. Homozygous deletions and mutations of the p16^(INK4)gene are frequent in human tumor cell lines. This evidence suggests thatthe p16^(INK4) gene is a tumor suppressor gene. This interpretation hasbeen challenged, however, by the observation that the frequency of thep16^(INK4) gene alterations is much lower in primary uncultured tumorsthan in cultured cell lines (Caldas et al., 1994; Cheng et al., 1994;Hussussian et al., 1994; Kamb et al., 1994; Kamb et al., 1994; Mori etal., 1994; Okamoto et al., 1994; Nobori et al., 1995; Orlow et al.,1994; Arap et al., 1995). Restoration of wild-type p16^(INK4) functionby transfection with a plasmid expression vector reduced colonyformation by some human cancer cell lines (Okamoto, 1994; Arap, 1995).

Other genes that may be employed according to the present inventioninclude Rb, APC, mda-7, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73,VHL, MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16 fusions, p21/p27fusions, anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras,myc, neu, raf, erb, fms, trk, ret, gsp, hst, abl, E1A, p300, genesinvolved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF,or their receptors) and MCC.

5. Regulators of Programmed Cell Death

Apoptosis, or programmed cell death, is an essential process for normalembryonic development, maintaining homeostasis in adult tissues, andsuppressing carcinogenesis (Kerr et al., 1972). The Bcl-2 family ofproteins and ICE-like proteases have been demonstrated to be importantregulators and effectors of apoptosis in other systems. The Bcl-2protein, discovered in association with follicular lymphoma, plays aprominent role in controlling apoptosis and enhancing cell survival inresponse to diverse apoptotic stimuli (Bakhshi et al., 1985; Cleary andSklar, 1985; Cleary et al., 1986; Tsujimoto et al., 1985; Tsujimoto andCroce, 1986). The evolutionarily conserved Bcl-2 protein now isrecognized to be a member of a family of related proteins, which can becategorized as death agonists or death antagonists.

Apo2 ligand (Apo2L, also called TRAIL) is a member of the tumor necrosisfactor (TNF) cytokine family. TRAIL activates rapid apoptosis in manytypes of cancer cells, yet is not toxic to normal cells. TRAIL mRNAoccurs in a wide variety of tissues. Most normal cells appear to beresistant to TRAIL's cytotoxic action, suggesting the existence ofmechanisms that can protect against apoptosis induction by TRAIL. Thefirst receptor described for TRAIL, called death receptor 4 (DR4),contains a cytoplasmic “death domain”; DR4 transmits the apoptosissignal carried by TRAIL. Additional receptors have been identified thatbind to TRAIL. One receptor, called DR5, contains a cytoplasmic deathdomain and signals apoptosis much like DR4. The DR4 and DR5 mRNAs areexpressed in many normal tissues and tumor cell lines. Recently, decoyreceptors such as DcR1 and DcR2 have been identified that prevent TRAILfrom inducing apoptosis through DR4 and DR5. These decoy receptors thusrepresent a novel mechanism for regulating sensitivity to apro-apoptotic cytokine directly at the cell's surface. The preferentialexpression of these inhibitory receptors in normal tissues suggests thatTRAIL may be useful as an anticancer agent that induces apoptosis incancer cells while sparing normal cells. (Marsters et al. 1999).

Subsequent to its discovery, it was shown that Bcl-2 acts to suppresscell death triggered by a variety of stimuli. Also, it now is apparentthat there is a family of Bcl-2 cell death regulatory proteins whichshare in common structural and sequence homologies. These differentfamily members have been shown to either possess similar functions toBcl-2 (e.g., BCl_(XL), Bcl_(W), Bcl_(S), Mcl-1, A1, Bfl-1) or counteractBcl-2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid,Bad, Harakiri). It is contemplated that any of these polypeptides,including TRAIL, or any other polypeptides that induce or promote ofapoptosis, may be operatively linked to a toxin, or that an antibodyrecognizing any of these polypeptides may also be attached to a toxin.

IV. Methods of Making Modified Proteins and Designer Toxins

The present invention encompasses methods of identifying antigenicregions on a protein, methods of identifying regions that are lessantigenic, methods of creating a less antigenic protein that possessesactivity that is comparable to a native protein, and methods of assayingand determining antigenicity and activity.

A. Antigenic Regions

A general discussion of antibodies and antibody detection methods can befound in previous sections. The term “antigenic region” refers to aportion of a protein that is specifically recognized by an antibody orT-cell receptor. The term “less antigenic” means that a protein orregion of a protein elicits a lower antibody response or is recognizedby fewer antibodies (polyclonal) or the binding association with anantibody is reduced.

Antigenicity is relative to a particular organism. In many of theembodiments of the present invention, the organism is a human, butantigenicity may be discussed with respect to other organisms as well,such as other mammals-monkeys, gorillas, cows, rabbits, mice, sheep,cats, dogs, pigs, goats, etc.—as well as avian organisms and any otherorganism that can elicit an immune response.

In some embodiments of the present invention, polyclonal sera isemployed with immunodetection methods previously discussed to identifyantigenic regions in a particular protein. Polyclonal sera may becollected from a variety of sources including workers suspected to havebeen occupationally exposed to a particular protein; patients suspectedof or diagnosed as having a condition or disease that is accompanied orcaused by the presence of antibodies to a particular protein ororganism; patients who no longer have been treated for a condition ordisease that is accompanied by the presence of antibodies to aparticular protein or organism; and random subjects.

B. Databases

In some methods of the present invention, protein databases are employedafter putative antigenic regions in a particular protein are identified.A region is then compared with a database containing protein sequencesfrom the organism in which a lower immune response against the region isdesired. A number of such databases exist both commercially andpublically, including GenBank, GenPept, SwissProt, PIR, PRF, PDB, all ofwhich are available from the National Center for BiotechnologyInformation website (http://wvw.ncbi.nlm.nih.gov/).

C. Removing and/or Replacing Antigenic Regions

Once an antigenic region is identified, it may be removed, creating atruncated protein. Alternatively, the region may be replaced with aregion believed to be less antigenic.

To remove the region, the polypeptide may be cleaved with proteinases,or a polynucleotide encoding the polypeptide may be manipulated toremove the antigenic region. The region may be removed from thepolynucleotide using conventional recombinant DNA technology, such asrestriction enzyme or DNAses.

The region may also be replaced with substitute amino acids. “Replaced”means that an amino acid at a particular position has been substitutedwith a different amino acid residue or with a modified amino acid. Thismay be accomplished by a number of ways. The region may be first removedand then the replacement region incorporated into a polynucleotide orthe polypeptide. Recombinant DNA technology may be used to incorporate aparticular coding region into a polynucleotide. Alternatively, anantigenic region may be mutagenized using site-specific mutagenesistechniques that are well known to those of ordinary skill in the art.

It is contemplated that amino acids flanking either side of an antigenicregion may also be removed or replaced, either to facilitate thecreation of a modified protein or to improve the protein in any way,such as decrease its antigenicity, increase the protein's stability,increase the activity of the protein, decrease the activity of theprotein, etc. Furthermore, multiple amino acids may be replaced orremoved from either antigenic region, flanking region, or both; thus,exactly or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320,330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460,470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600,620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880,900, 920, 940, 960, 980, 1000, or more amino acids may be removed orreplaced.

Assays to determine antigenicity or activity of a modified protein aredescribed herein, for example, in a section describing immunodetectionmethods, or they are well known to those of skill in the art.Appropriate assays for a particular protein will vary depending on theprotein. Enzymatic assays may be appropriate to evaluate the activity ofan enzyme, for example. One of skill in the art would be able toevaluate the activity of a modified protein relative to the nativeprotein. As discussed above, a modified protein may be attached(conjugated or fused) to another polypeptide, peptide, or protein. Oneof skill in the art would also be able to evaluate any modifiedconjugated or fusion protein of the invention depending upon theactivity or activities of the polypeptide components.

V. Combination Therapies

In order to increase the efficacy of any of the therapeutic compositionsof the present invention, it may be desirable to combine thesecompositions with other agents effective in the treatment of aparticular disease or condition. It is contemplated that a wide varietyof conditions or diseases may be treated, such as microbialpathogenesis, AIDS, autoimmune diseases, hyperproliferative disordersincluding cancers, leukemias, arthritis, inflammatory diseases,cardiovascular diseases and conditions, pathogenic diseases andconditions, and diabetes. The treatment of AIDS, cancer, and otherhyperproliferative disorders is specifically contemplated. Variouscombinations of therapies may be employed as such, in which acomposition comprising a modified protein is “A” and the secondary agentis “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/AA. Treatment of Hyperproliferative Diseases

Hyperproliferative diseases include cancer, for which there is a widevariety of treatment regimens such as anti-cancer agents or surgery. An“anti-cancer” agent is capable of negatively affecting cancer in asubject, for example, by killing cancer cells, inducing apoptosis incancer cells, reducing the growth rate of cancer cells, reducing theincidence or number of metastases, reducing tumor size, inhibiting tumorgrowth, reducing the blood supply to a tumor or cancer cells, promotingan immune response against cancer cells or a tumor, preventing orinhibiting the progression of cancer, or increasing the lifespan of asubject with cancer.

Anti-cancer agents include biological agents (biotherapy), chemotherapyagents, and radiotherapy agents. More generally, these othercompositions would be provided in a combined amount effective to kill orinhibit proliferation of the cell. This process may involve contactingthe cells with the expression construct and the agent(s) or multiplefactor(s) at the same time. This may be achieved by contacting the cellwith a single composition or pharmacological formulation that includesboth agents, or by contacting the cell with two distinct compositions orformulations, at the same time, wherein one composition includes theexpression construct and the other includes the second agent(s).

Tumor cell resistance to chemotherapy and radiotherapy agents representsa major problem in clinical oncology. One goal of current cancerresearch is to find ways to improve the efficacy of chemo- andradiotherapy by combining it with gene therapy. For example, the herpessimplex-thymidine kinase (HS-tK) gene, when delivered to brain tumors bya retroviral vector system, successfully induced susceptibility to theantiviral agent ganciclovir (Culver, et al., 1992). In the context ofthe present invention, it is contemplated that therapy with modifiedproteins could be used similarly in conjunction with chemotherapeutic,radiotherapeutic, immunotherapeutic or other biological intervention, inaddition to other pro-apoptotic or cell cycle regulating agents.

Alternatively, the gene therapy or protein administration of modifiedproteins may precede or follow the other agent treatment by intervalsranging from minutes to weeks. In embodiments where the other agent andexpression construct are applied separately to the cell, one wouldgenerally ensure that a significant period of time did not expirebetween the time of each delivery, such that the agent and expressionconstruct would still be able to exert an advantageously combined effecton the cell. In such instances, it is contemplated that one may contactthe cell with both modalities within about 12-24 h of each other and,more preferably, within about 6-12 h of each other. In some situations,it may be desirable to extend the time period for treatmentsignificantly, however, where several d (2, 3, 4, 5, 6 or 7) to severalwk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

Administration of the therapeutic expression constructs of the presentinvention to a patient will follow general protocols for theadministration of chemotherapeutics, taking into account the toxicity,if any, of the vector. It is expected that the treatment cycles would berepeated as necessary. It also is contemplated that various standardtherapies, as well as surgical intervention, may be applied incombination with the described hyperproliferative cell therapy.

a. Chemotherapy

Cancer therapies also include a variety of combination therapies withboth chemical and radiation based treatments. Combination chemotherapiesinclude, for example, cisplatin (CDDP), carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16),tamoxifen, raloxifene, estrogen receptor binding agents, taxol,gemcitabien, navelbine, farnesyl-protein transferase inhibitors,transplatinum, 5-fluorouracil, vincristine, vinblastine andmethotrexate, Temazolomide (an aqueous form of DTIC), or any analog orderivative variant of the foregoing. The combination of chemotherapywith biological therapy is known as biochemotherapy.

In some embodiments of the present invention, it is contemplated that achemotherapeutic is operatively attached to a modified protein, such asa toxin molecule.

b. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageon DNA, on the precursors of DNA, on the replication and repair of DNA,and on the assembly and maintenance of chromosomes. Dosage ranges forX-rays range from daily doses of 50 to 200 roentgens for prolongedperiods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.Dosage ranges for radioisotopes vary widely, and depend on the half-lifeof the isotope, the strength and type of radiation emitted, and theuptake by the neoplastic cells.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

c. Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Asdiscussed above with respect to claimed compositions, the antibody alsomay be conjugated to a drug or toxin (chemotherapeutic, radionuclide,ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely asa targeting agent. Alternatively, the effector may be a lymphocytecarrying a surface molecule that interacts, either directly orindirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells. The combination of therapeuticmodalities, i.e., direct cytotoxic activity and immune activation mayprovide therapeutic benefit in the treatment of cancer, and thus, it iscontemplated that immunotherapeutics may be used in conjunction with anytherapeutic composition of the invention. For example, two differentimmunotoxins may be administered to a subject or an immunotoxin may beadministered in combination with another immunotherapeutic compound inthe treatment of a disease, such as cancer.

In one aspect of immunotherapy, the tumor cell must bear some markerthat is amenable to targeting, i.e., is not present on the majority ofother cells. Many tumor markers exist and any of these may be suitablefor targeting in the context of the present invention, as discussedabove. An alternative aspect of immunotherapy is to combine apro-apoptotic effect with immune stimulatory effects. However, alternateimmune stimulating molecules also exist including: cytokines such asIL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1,IL-8 and growth factors such as FLT3 ligand. Combining immunestimulating molecules, either as proteins or using gene delivery incombination with an immunotoxin directed again a tumor may enhanceanti-tumor effects.

As discussed earlier, examples of immunotherapies currently underinvestigation or in use are immune adjuvants (e.g., Mycobacterium bovis,Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds)(U.S. Pat. No. 5,801,005; U.S. Pat. No. 5,739,169; Hui and Hashimoto,1998; Christodoulides et al., 1998), cytokine therapy (e.g., interferonsα, β and γ; IL-1, GM-CSF and TNF) (Bukowski et al., 1998; Davidson etal., 1998; Hellstrand et al., 1998) gene therapy (e.g., TNF, IL-1, IL-2,p53) (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. No.5,830,880 and U.S. Pat. No. 5,846,945) and monoclonal antibodies (e.g.,anti-ganglioside GM2, anti-HER-2, anti-p185) (Pietras et al., 1998;Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311). Herceptin(trastuzumab) is a chimeric (mouse-human) monoclonal antibody thatblocks the HER2-neu receptor. It possesses anti-tumor activity and hasbeen approved for use in the treatment of malignant tumors (Dillman,1999).

i. Passive Immunotherapy

A number of different approaches for passive immunotherapy of cancerexist. They may be broadly categorized into the following: injection ofantibodies alone; injection of antibodies coupled to toxins orchemotherapeutic agents; injection of antibodies coupled to radioactiveisotopes; injection of anti-idiotype antibodies; and finally, purging oftumor cells in bone marrow.

Preferably, human monoclonal antibodies are employed in passiveimmunotherapy, as they produce few or no side effects in the patient.However, their application is somewhat limited by their scarcity andhave so far only been administered intralesionally. Human monoclonalantibodies to ganglioside antigens have been administeredintralesionally to patients suffering from cutaneous recurrent melanoma(Irie & Morton, 1986). Regression was observed in six out of tenpatients, following, daily or weekly, intralesional injections. Inanother study, moderate success was achieved from intralesionalinjections of two human monoclonal antibodies (Irie et al., 1989).

It may be favorable to administer more than one monoclonal antibodydirected against two different antigens or even antibodies with multipleantigen specificity. Treatment protocols also may include administrationof lymphokines or other immune enhancers as described by Bajorin et al.(1988). The development of human monoclonal antibodies is described infurther detail elsewhere in the specification.

ii. Active Immunotherapy

In active immunotherapy, an antigenic peptide, polypeptide or protein,or an autologous or allogenic tumor cell composition or “vaccine” isadministered, generally with a distinct bacterial adjuvant (Ravindranath& Morton, 1991; Morton & Ravindranath, 1996; Morton et al., 1992;Mitchell et al., 1990; Mitchell et al., 1993). In melanomaimmunotherapy, those patients who elicit high IgM response often survivebetter than those who elicit no or low IgM antibodies (Morton et al.,1992). IgM antibodies are often transient antibodies and the exceptionto the rule appears to be anti-ganglioside or anticarbohydrateantibodies.

iii. Adoptive Immunotherapy

In adoptive immunotherapy, the patient's circulating lymphocytes, ortumor infiltrated lymphocytes, are isolated in vitro, activated bylymphokines such as IL-2 or transduced with genes for tumor necrosis,and readministered (Rosenberg et al., 1988; 1989). To achieve this, onewould administer to an animal, or human patient, an immunologicallyeffective amount of activated lymphocytes in combination with anadjuvant-incorporated antigenic peptide composition as described herein.The activated lymphocytes will most preferably be the patient's owncells that were earlier isolated from a blood or tumor sample andactivated (or “expanded”) in vitro. This form of immunotherapy hasproduced several cases of regression of melanoma and renal carcinoma,but the percentage of responders were few compared to those who did notrespond.

d. Genes

In yet another embodiment, the secondary treatment is gene therapy inwhich a therapeutic polynucleotide is administered before, after, or atthe same time as a modified polypeptide or a polynucleotide encoding amodified polypeptide. Alternatively, a single vector encoding twodifferent therapeutic polypeptide molecules may be used. A variety ofproteins are encompassed within the invention, some of which aredescribed earlier. For example, gene therapy may be employed withrespect to providing a wild-type tumor suppressor gene to a cancer cell.

e. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and microscopically controlled surgery(Mohs' surgery). It is further contemplated that the present inventionmay be used in conjunction with removal of superficial cancers,precancers, or incidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

f. Other Agents

It is contemplated that other agents may be used in combination with thepresent invention to improve the therapeutic efficacy of treatment.These additional agents include immunomodulatory agents, agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion,agents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers, or other biological agents. Immunomodulatory agentsinclude tumor necrosis factor; interferon alpha, beta, and gamma; IL-2and other cytokines; F42K and other cytokine analogs; or MIP-1,MIP-1beta, MCP-1, RANTES, and other chemokines. It is furthercontemplated that the upregulation of cell surface receptors or theirligands such as Fas/Fas ligand, DR4 or DR5/TRAIL (Apo-2 ligand) wouldpotentiate the anti-cancer abilities of the present invention byestablishment of an autocrine or paracrine effect on hyperproliferativecells. Increases intercellular signaling by elevating the number of GAPjunctions would increase the anti-hyperproliferative effects on theneighboring hyperproliferative cell population. In other embodiments,cytostatic or differentiation agents can be used in combination with thepresent invention to improve the anti-hyperproliferative efficacy of thetreatments. Inhibitors of cell adhesion are contemplated to improve theefficacy of the present invention. Examples of cell adhesion inhibitorsare focal adhesion kinase (FAKs) inhibitors and Lovastatin. It isfurther contemplated that other agents that increase the sensitivity ofa hyperproliferative cell to apoptosis, such as the antibody c225, couldbe used in combination with the present invention to improve thetreatment efficacy.

There have been many advances in the therapy of cancer following theintroduction of cytotoxic chemotherapeutic drugs. However, one of theconsequences of chemotherapy is the development/acquisition ofdrug-resistant phenotypes and the development of multiple drugresistance. The development of drug resistance remains a major obstaclein the treatment of such tumors and therefore, there is an obvious needfor alternative approaches such as gene therapy.

Studies from a number of investigators have demonstrated that tumorcells that are resistant to TRAIL can be sensitized by subtoxicconcentrations of drugs/cytokines and the sensitized tumor cells aresignificantly killed by TRAIL. (Bonavida et al., 1999; Bonavida et al,2000; Gliniak et al., 1999; Keane et al., 1999). Furthermore, thecombination of chemotherapeutics, such as CPT-11 or doxorubicin, withTRAIL also lead to enhanced anti-tumor activity and an increase inapoptosis. Some of these effects may be mediated via up-regulation ofTRAIL or cognate receptors, whereas others may not.

Another form of therapy for use in conjunction with chemotherapy,radiation therapy or biological therapy includes hyperthermia, which isa procedure in which a patient's tissue is exposed to high temperatures(up to 106° F.). External or internal heating devices may be involved inthe application of local, regional, or whole-body hyperthermia. Localhyperthermia involves the application of heat to a small area, such as atumor. Heat may be generated externally with high-frequency wavestargeting a tumor from a device outside the body. Internal heat mayinvolve a sterile probe, including thin, heated wires or hollow tubesfilled with warm water, implanted microwave antennae, or radiofrequencyelectrodes.

A patient's organ or a limb is heated for regional therapy, which isaccomplished using devices that produce high energy, such as magnets.Alternatively, some of the patient's blood may be removed and heatedbefore being perfused into an area that will be internally heated.Whole-body heating may also be implemented in cases where cancer hasspread throughout the body. Warm-water blankets, hot wax, inductivecoils, and thermal chambers may be used for this purpose.

Hormonal therapy may also be used in conjunction with the presentinvention or in combination with any other cancer therapy previouslydescribed. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen. This treatment is often used in combinationwith at least one other cancer therapy as a treatment option or toreduce the risk of metastases.

B. Viral Pathogenesis

Of course it is understood that compositions and methods of the presentinvention have relevance to the treatment or diagnosis of viralpathogenesis. For example, it is contemplated that the invention may beused for the treatment of AIDS, which is caused by HIV infection.Therefore, the present invention may be used in combination with theadministration of traditional therapies. Some such therapies aredescribed below.

1. AZT

A well-known, traditional therapy for the treatment of AIDS involveszovidovudine (AZT™ available from Burroughs Wellcome). This is one of aclass of nucleoside analogues known as dideoxynucleosides which blockHIV replication by inhibiting HIV reverse transcriptase. The anti-AIDSdrug zidovudine (also known as AZT) may also be used in limitedcircumstances, mostly in combination with rifampin, as described byBurger et al. (1993).

The compositions and methods disclosed herein will be particularlyeffective in conjunction with other forms of therapy, such as AZT and/orprotease inhibitors that are designed to inhibit viral replication, bymaintaining desirable levels of white blood cells. This, in effect, buysthe patient the time necessary for the anti-viral therapies to work.

2. HAART

New combination drug therapy has shown promising results in thetreatment of HIV-infected patients. Treatment with potent anti-HIV drugcombinations is referred to as “highly active antiretroviral therapy”(HAART), and it has provided clinical improvement, longer survival, andimproved quality of life for people infected with HIV during all fourstages of HIV disease. Examples of HAART include a protease inhibitor(indinavir, nelfinavir, ritonavir, ritonavir/saquinavir, or saquinavir)combined with two nucleoside analogs (AZT/ddI, d4T/ddI, AZT/ddC,AZT/3TC, or d4T/3TC).

V. Pharmaceutical Compositions and Routes of Administration

The present invention contemplates nucleic acid molecules encodingmodified proteins (including fusion proteins), as well as modifiedproteins that may be conjugated to another proteinaceous compound or toa small molecule. In some embodiments, pharmaceutical compositions areadministered to a subject. Different aspects of the present inventioninvolve administering an effective amount of an aqueous composition. Inanother embodiment of the present invention, modified gelonin as animmunotoxin is specifically contemplated. Such compositions willgenerally be dissolved or dispersed in a pharmaceutically acceptablecarrier or aqueous medium. Additionally, such compounds can beadministered in combination with another treatment depending upon thedisease or condition being treated. Treatment of AIDS could includeadministration of HAART or of AZT, or both, while treatment of cancercould include surgery or the administration of chemotherapy,radiotherapy, immunotherapy, or hormones.

A. Routes of Administration

Those of skill in the art are well aware of how to apply gene deliveryto in vivo and ex vivo situations. For viral vectors, one generally willprepare a viral vector stock. Depending on the kind of virus and thetiter attainable, one will deliver 1 to 100, 10 to 50, 100-1000, or upto 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, or 1×10¹²infectious viral particles to the patient. Similar figures may beextrapolated for liposomal or other non-viral formulations by comparingrelative uptake efficiencies. Formulation as a pharmaceuticallyacceptable composition is discussed below.

The phrases “pharmaceutically acceptable” or “pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce an adverse, allergic, or other untoward reaction whenadministered to an animal, or human, as appropriate. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like. The use of suchmedia and agents for pharmaceutical active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredients, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients, such asother anti-cancer agents, can also be incorporated into thecompositions.

In addition to the compounds formulated for parenteral administration,such as those for intravenous or intramuscular injection, otherpharmaceutically acceptable forms include, e.g., tablets or other solidsfor oral administration; time release capsules; and any other formcurrently used, including cremes, lotions, mouthwashes, inhalants andthe like.

The active compounds of the present invention can be formulated forparenteral administration, e.g., formulated for injection via theintravenous, intramuscular, intrathoracic, subcutaneous, or evenintraperitoneal routes. The preparation of an aqueous composition thatcontains a compound or compounds that increase the expression of an MHCclass I molecule will be known to those of skill in the art in light ofthe present disclosure. Typically, such compositions can be prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for use to prepare solutions or suspensions upon the additionof a liquid prior to injection can also be prepared; and, thepreparations can also be emulsified.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil, or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that it may be easily injected. It also should be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi.

The active compounds may be formulated into a composition in a neutralor salt form. Pharmaceutically acceptable salts, include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

The carrier also can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion, and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques, which yield a powder of the active ingredient, plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

In certain cases, the therapeutic formulations of the invention also maybe prepared in forms suitable for topical administration, such as incremes and lotions. These forms may be used for treating skin-associateddiseases, such as various sarcomas.

Administration of therapeutic compositions according to the presentinvention will be via any common route so long as the target tissue isavailable via that route. In cases where the present invention is usedas a viral vector, a primary consideration will be the desired locationfor the heterologous sequences carried by the vector. Routes ofadministration include oral, nasal, buccal, rectal, vaginal or topical.For example, topical administration would be particularly advantageousfor treatment of melanoma or AIDS-related skin conditions, or where aheterologous gene useful in treating a skin condition is carried by aviral vector. Alternatively, administration will be by orthotopic,intradermal subcutaneous, intramuscular, intraperitoneal or intravenousinjection. Such compositions would normally be administered aspharmaceutically acceptable compositions that include physiologicallyacceptable carriers, buffers or other excipients. For treatment ofconditions of the lungs, aerosol delivery to the lung is contemplated.Volume of the aerosol is between about 0.01 ml and 0.5 ml. Similarly, apreferred method for treatment of colon-associated disease would be viaenema. Volume of the enema is between about 1 ml and 100 ml. Directintratumoral injection is the preferred mode, with continuousintratumoral perfusion a more specific embodiment.

In certain embodiments, it may be desirable to provide a continuoussupply of therapeutic compositions to the patient. For intravenous orintraarterial routes, this is accomplished by drip system. For topicalapplications, repeated application would be employed. For variousapproaches, delayed release formulations could be used that providedlimited but constant amounts of the therapeutic agent over and extendedperiod of time. For internal application, continuous perfusion, forexample with a viral vector carrying a heterologous nucleic acidsegment, of the region of interest may be preferred. This could beaccomplished by catheterization, post-operatively in some cases,followed by continuous administration of the therapeutic agent. The timeperiod for perfusion would be selected by the clinician for theparticular patient and situation, but times could range from about 1-2hours, to 2-6 hours, to about 6-10 hours, to about 10-24 hours, to about1-2 days, to about 1-2 weeks or longer. Generally, the dose of thetherapeutic composition via continuous perfusion will be equivalent tothat given by single or multiple injections, adjusted for the period oftime over which the injections are administered. It is believed thathigher doses may be achieved via perfusion, however.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 mL of isotonic NaCi solutionand either added to 1000 mL of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, Remington's PharmaceuticalSciences, 1990). Some variation in dosage will necessarily occurdepending on the condition of the subject being treated. The personresponsible for administration will, in any event, determine theappropriate dose for the individual subject.

An effective amount of the therapeutic composition is determined basedon the intended goal. The term “unit dose” or “dosage” refers tophysically discrete units suitable for use in a subject, each unitcontaining a predetermined-quantity of the therapeutic compositioncalculated to produce the desired responses, discussed above, inassociation with its administration, i.e., the appropriate route andtreatment regimen. The quantity to be administered, both according tonumber of treatments and unit dose, depends on the protection desired.

Precise amounts of the therapeutic composition also depend on thejudgment of the practitioner and are peculiar to each individual.Factors affecting dose include physical and clinical state of thepatient, the route of administration, the intended goal of treatment(alleviation of symptoms versus cure) and the potency, stability, andtoxicity of the particular therapeutic substance.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

As used herein, the term in vitro administration refers to manipulationsperformed on cells removed from an animal, including, but not limitedto, cells in culture. The term ex vivo administration refers to cellsthat have been manipulated in vitro, and are subsequently administeredto a living animal. The term in vivo administration includes allmanipulations performed on cells within an animal.

In certain aspects of the present invention, the compositions may beadministered either in vitro, ex vivo, or in vivo. In certain in vitroembodiments, an expression construct encoding a modified protein may betransduced into a host cell. The transduced cells can then be used forin vitro analysis, or alternatively for in vivo administration.

U.S. Pat. Nos. 4,690,915 and 5,199,942, both incorporated herein byreference, disclose methods for ex vivo manipulation of bloodmononuclear cells and bone marrow cells for use in therapeuticapplications.

In vivo administration of the compositions of the present invention arealso contemplated. Examples include, but are not limited to,transduction of bladder epithelium by administration of the transducingcompositions of the present invention through intravesiclecatheterization into the bladder (Bass, 1995), and transduction of livercells by infusion of appropriate transducing compositions through theportal vein via a catheter (Bao, 1996). Additional examples includedirect injection of tumors with the instant transducing compositions,and either intranasal or intratracheal (Dong, 1996) instillation oftransducing compositions to effect transduction of lung cells.

The present invention can be administered intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, rectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,intravesicularlly, mucosally, intrapericardially, orally, topically,locally and/or using aerosol, injection, infusion, continuous infusion,localized perfusion bathing target cells directly or via a catheterand/or lavage.

B. Lipid Compositions

In certain embodiments, the present invention concerns a novelcomposition comprising one or more lipids associated with apolynucleotide or polypeptide of the claimed invention. A lipid is asubstance that is characteristically insoluble in water and extractablewith an organic solvent. Compounds than those specifically describedherein are understood by one of skill in the art as lipids, and areencompassed by the compositions and methods of the present invention.

A lipid may be naturally occurring or synthetic (i.e., designed orproduced by man). However, a lipid is usually a biological substance.Biological lipids are well known in the art, and include for example,neutral fats, phospholipids, phosphoglycerides, steroids, terpenes,lysolipids, glycosphingolipids, glucolipids, sulphatides, lipids withether and ester-linked fatty acids and polymerizable lipids, andcombinations thereof.

1. Lipid Types

A neutral fat may comprise a glycerol and a fatty acid. A typicalglycerol is a three carbon alcohol. A fatty acid generally is a moleculecomprising a carbon chain with an acidic moiety (e.g., carboxylic acid)at an end of the chain. The carbon chain may of a fatty acid may be ofany length, however, it is preferred that the length of the carbon chainbe of from about 2, about 3, about 4, about 5, about 6, about 7, about8, about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22,about 23, about 24, about 25, about 26, about 27, about 28, about 29, toabout 30 or more carbon atoms, and any range derivable therein. However,a preferred range is from about 14 to about 24 carbon atoms in the chainportion of the fatty acid, with about 16 to about 18 carbon atoms beingparticularly preferred in certain embodiments. In certain embodimentsthe fatty acid carbon chain may comprise an odd number of carbon atoms,however, an even number of carbon atoms in the chain may be preferred incertain embodiments. A fatty acid comprising only single bonds in itscarbon chain is called saturated, while a fatty acid comprising at leastone double bond in its chain is called unsaturated.

Specific fatty acids include, but are not limited to, linoleic acid,oleic acid, palmitic acid, linolenic acid, stearic acid, lauric acid,myristic acid, arachidic acid, palmitoleic acid, arachidonic acidricinoleic acid, tuberculosteric acid, lactobacillic acid. An acidicgroup of one or more fatty acids is covalently bonded to one or morehydroxyl groups of a glycerol. Thus, a monoglyceride comprises aglycerol and one fatty acid, a diglyceride comprises a glycerol and twofatty acids, and a triglyceride comprises a glycerol and three fattyacids.

A phospholipid generally comprises either glycerol or an sphingosinemoiety, an ionic phosphate group to produce an amphipathic compound, andone or more fatty acids. Types of phospholipids include, for example,phophoglycerides, wherein a phosphate group is linked to the firstcarbon of glycerol of a diglyceride, and sphingophospholipids (e.g.,sphingomyelin), wherein a phosphate group is esterified to a sphingosineamino alcohol. Another example of a sphingophospholipid is a sulfatide,which comprises an ionic sulfate group that makes the moleculeamphipathic. A phopholipid may, of course, comprise further chemicalgroups, such as for example, an alcohol attached to the phosphate group.Examples of such alcohol groups include serine, ethanolamine, choline,glycerol and inositol. Thus, specific phosphoglycerides include aphosphatidyl serine, a phosphatidyl ethanolamine, a phosphatidylcholine, a phosphatidyl glycerol or a phosphotidyl inositol. Otherphospholipids include a phosphatidic acid or a diacetyl phosphate. Inone aspect, a phosphatidylcholine comprises adioleoylphosphatidylcholine (a.k.a. cardiolipin), an eggphosphatidylcholine, a dipalmitoyl phosphalidycholine, a monomyristoylphosphatidylcholine, a monopalmitoyl phosphatidylcholine, a monostearoylphosphatidylcholine, a monooleoyl phosphatidylcholine, a dibutroylphosphatidylcholine, a divaleroyl phosphatidylcholine, a dicaproylphosphatidylcholine, a diheptanoyl phosphatidylcholine, a dicapryloylphosphatidylcholine or a distearoyl phosphatidylcholine.

A glycolipid is related to a sphinogophospholipid, but comprises acarbohydrate group rather than a phosphate group attached to a primaryhydroxyl group of the sphingosine. A type of glycolipid called acerebroside comprises one sugar group (e.g., a glucose or galactose)attached to the primary hydroxyl group. Another example of a glycolipidis a ganglioside (e.g., a monosialoganglioside, a GM1), which comprisesabout 2, about 3, about 4, about 5, about 6, to about 7 or so sugargroups, that may be in a branched chain, attached to the primaryhydroxyl group. In other embodiments, the glycolipid is a ceramide(e.g., lactosylceramide).

A steroid is a four-membered ring system derivative of a phenanthrene.Steroids often possess regulatory functions in cells, tissues andorganisms, and include, for example, hormones and related compounds inthe progestagen (e.g., progesterone), glucocoricoid (e.g., cortisol),mineralocorticoid (e.g., aldosterone), androgen (e.g., testosterone) andestrogen (e.g., estrone) families. Cholesterol is another example of asteroid, and generally serves structural rather than regulatoryfunctions. Vitamin D is another example of a sterol, and is involved incalcium absorption from the intestine.

A terpene is a lipid comprising one or more five carbon isoprene groups.Terpenes have various biological functions, and include, for example,vitamin A, coenyzme Q and carotenoids (e.g., lycopene and β-carotene).

2. Charged and Neutral Lipid Compositions

In certain embodiments, a lipid component of a composition is unchargedor primarily uncharged. In one embodiment, a lipid component of acomposition comprises one or more neutral lipids. In another aspect, alipid component of a composition may be substantially free of anionicand cationic lipids, such as certain phospholipids and cholesterol. Incertain aspects, a lipid component of tan uncharged or primarilyuncharged lipid composition comprises about 95%, about 96%, about 97%,about 98%, about 99% or 100% lipids without a charge, substantiallyuncharged lipid(s), and/or a lipid mixture with equal numbers ofpositive and negative charges.

In other aspects, a lipid composition may be charged. For example,charged phospholipids may be used for preparing a lipid compositionaccording to the present invention and can carry a net positive chargeor a net negative charge. In a non-limiting example, diacetyl phosphatecan be employed to confer a negative charge on the lipid composition,and stearylamine can be used to confer a positive charge on the lipidcomposition.

3. Making Lipids

Lipids can be obtained from natural sources, commercial sources orchemically synthesized, as would be known to one of ordinary skill inthe art. For example, phospholipids can be from natural sources, such asegg or soybean phosphatidylcholine, brain phosphatidic acid, brain orplant phosphatidylinositol, heart cardiolipin and plant or bacterialphosphatidylethanolamine. In another example, lipids suitable for useaccording to the present invention can be obtained from commercialsources. For example, dimyristyl phosphatidylcholine (“DMPC”) can beobtained from Sigma Chemical Co., dicetyl phosphate (“DCP”) is obtainedfrom K & K Laboratories (Plainview, N.Y.); cholesterol (“Chol”) isobtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol(“DDMPG”) and other lipids may be obtained from Avanti Polar Lipids,Inc. (Birmingham, Ala.). In certain embodiments, stock solutions oflipids in chloroform or chloroform/methanol can be stored at about −20°C. Preferably, chloroform is used as the only solvent since it is morereadily evaporated than methanol.

4. Lipid Composition Structures

A compound associated with a lipid may be dispersed in a solutioncontaining a lipid, dissolved with a lipid, emulsified with a lipid,mixed with a lipid, combined with a lipid, covalently bonded to a lipid,contained as a suspension in a lipid or otherwise associated with alipid. A lipid or lipid-associated composition of the present inventionis not limited to any particular structure. For example, they may alsosimply be interspersed in a solution, possibly forming aggregates whichare not uniform in either size or shape. In another example, they may bepresent in a bilayer structure, as micelles, or with a “collapsed”structure. In another non-limiting example, a lipofectamine(Gibco BRL)or Superfect (Qiagen) complex is also contemplated.

In certain embodiments, a lipid composition may comprise about 1%, about2%, about 3%, about 4% about 5%, about 6%, about 7%, about 8%, about 9%,about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%,about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%,about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%,about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%,about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%,or any range derivable therein, of a particular lipid, lipid type ornon-lipid component such as a drug, protein, sugar, nucleic acids orother material disclosed herein or as would be known to one of skill inthe art. In a non-limiting example, a-lipid composition may compriseabout 10% to about 20% neutral lipids, and about 33% to about 34% of acerebroside, and about 1% cholesterol. In another non-limiting example,a liposome may comprise about 4% to about 12% terpenes, wherein about 1%of the micelle is specifically lycopene, leaving about 3% to about 11%of the liposome as comprising other terpenes; and about 10% to about 35%phosphatidyl choline, and about 1% of a drug. Thus, it is contemplatedthat lipid compositions of the present invention may comprise any of thelipids, lipid types or other components in any combination or percentagerange.

a. Emulsions

A lipid may be comprised in an emulsion. A lipid emulsion is asubstantially permanent heterogenous liquid mixture of two or moreliquids that do not normally dissolve in each other, by mechanicalagitation or by small amounts of additional substances known asemulsifiers. Methods for preparing lipid emulsions and adding additionalcomponents are well known in the art (e.g., Modem Pharmaceutics, 1990,incorporated herein by reference).

For example, one or more lipids are added to ethanol or chloroform orany other suitable organic solvent and agitated by hand or mechanicaltechniques. The solvent is then evaporated from the mixture leaving adried glaze of lipid. The lipids are resuspended in aqueous media, suchas phosphate buffered saline, resulting in an emulsion. To achieve amore homogeneous size distribution of the emulsified lipids, the mixturemay be sonicated using conventional sonication techniques, furtheremulsified using microfluidization (using, for example, aMicrofluidizer, Newton, Mass.), and/or extruded under high pressure(such as, for example, 600 psi) using an Extruder Device (LipexBiomembranes, Vancouver, Canada).

b. Micelles

A lipid may be comprised in a micelle. A micelle is a cluster oraggregate of lipid compounds, generally in the form of a lipidmonolayer, and may be prepared using any micelle producing protocolknown to those of skill in the art (e.g., Canfield et al., 1990;El-Gorab et al, 1973; Colloidal Surfactant, 1963; and Catalysis inMicellar and Macromolecular Systems, 1975, each incorporated herein byreference). For example, one or more lipids are typically made into asuspension in an organic solvent, the solvent is evaporated, the lipidis resuspended in an aqueous medium, sonicated and then centrifuged.

5. Liposomes

In particular embodiments, a lipid comprises a liposome. A “liposome” isa generic term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes may be characterized as having vesicularstructures with a bilayer membrane, generally comprising a phospholipid,and an inner medium that generally comprises an aqueous composition.

A multilamellar liposome has multiple lipid layers separated by aqueousmedium. They form spontaneously when lipids comprising phospholipids aresuspended in an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Lipophilic molecules or molecules with lipophilicregions may also dissolve in or associate with the lipid bilayer.

In specific aspects, a lipid and/or modified protein or polynucleotideencoding a modified protein may be, for example, encapsulated in theaqueous interior of a liposome, interspersed within the lipid bilayer ofa liposome, attached to a liposome via a linking molecule that isassociated with both the liposome and the composition, entrapped in aliposome, complexed with a liposome, etc.

a. Making Liposomes

A liposome used according to the present invention can be made bydifferent methods, as would be known to one of ordinary skill in theart.

For example, a phospholipid (Avanti Polar Lipids, Alabaster, Ala.), suchas for example the neutral phospholipid dioleoylphosphatidylcholine(DOPC), is dissolved in tert-butanol. The lipid(s) is then mixed withthe polynucleotide or polypeptide, and/or other component(s). Tween 20is added to the lipid mixture such that Tween 20 is about 5% of thecomposition's weight. Excess tert-butanol is added to this mixture suchthat the volume of tert-butanol is at least 95%. The mixture isvortexed, frozen in a dry ice/acetone bath and lyophilized overnight.The lyophilized preparation is stored at −20° C. and can be used up tothree months. When required the lyophilized liposomes are reconstitutedin 0.9% saline. The average diameter of the particles obtained usingTween 20 for encapsulating the compound is about 0.7 to about 1.0 μm indiameter.

Alternatively, a liposome can be prepared by mixing lipids in a solventin a container, e.g., a glass, pear-shaped flask. The container shouldhave a volume ten-times greater than the volume of the expectedsuspension of liposomes. Using a rotary evaporator, the solvent isremoved at approximately 40° C. under negative pressure. The solventnormally is removed within about 5 min. to 2 hours, depending on thedesired volume of the liposomes. The composition can be dried further ina desiccator under vacuum. The dried lipids generally are discardedafter about 1 week because of a tendency to deteriorate with time.

Dried lipids can be hydrated at approximately 25-50 mM phospholipid insterile, pyrogen-free water by shaking until all the lipid film isresuspended. The aqueous liposomes can be then separated into aliquots,each placed in a vial, lyophilized and sealed under vacuum.

In other alternative methods, liposomes can be prepared in accordancewith other known laboratory procedures (e.g., see Bangham et al., 1965;Gregoriadis, 1979; Deamer and Uster 1983, Szoka and Papahadjopoulos,1978, each incorporated herein by reference in relevant part). Thesemethods differ in their respective abilities to entrap aqueous materialand their respective aqueous space-to-lipid ratios.

The dried lipids or lyophilized liposomes prepared as described abovemay be dehydrated and reconstituted in a solution of inhibitory peptideand diluted to an appropriate concentration with an suitable solvent,e.g., DPBS. The mixture is then vigorously shaken in a vortex mixer.Unencapsulated additional materials, such as agents including but notlimited to hormones, drugs, nucleic acid constructs and the like, areremoved by centrifugation at 29,000×g and the liposomal pellets washed.The washed liposomes are resuspended at an appropriate totalphospholipid concentration, e.g., about 50-200 mM. The amount ofadditional material or active agent encapsulated can be determined inaccordance with standard methods. After determination of the amount ofadditional material or active agent encapsulated in the liposomepreparation, the liposomes may be diluted to appropriate concentrationsand stored at 4° C. until use. A pharmaceutical composition comprisingthe liposomes will usually include a sterile, pharmaceuticallyacceptable carrier or diluent, such as water or saline solution.

The size of a liposome varies depending on the method of synthesis.Liposomes in the present invention can be a variety of sizes. In certainembodiements, the liposomes are small, e.g., less than about 100 nm,about 90 nm, about 80 nm, about 70 nm, about 60 nm, or less than about50 nm in external diameter. In preparing such liposomes, any protocoldescribed herein, or as would be known to one of ordinary skill in theart may be used. Additional non-limiting examples of preparing liposomesare described in U.S. Pat. Nos. 4,728,578, 4,728,575, 4,737,323,4,533,254, 4,162,282, 4,310,505, and 4,921,706; InternationalApplications PCT/US85/01161 and PCT/US89/05040; U.K. Patent ApplicationGB 2193095 A; Mayer et al., 1986; Hope et al., 1985; Mayhew et al. 1987;Mayhew et al., 1984; Cheng et al., 1987; and Liposome Technology, 1984,each incorporated herein by reference).

A liposome suspended in an aqueous solution is generally in the shape ofa spherical vesicle, having one or more concentric layers of lipidbilayer molecules. Each layer consists of a parallel array of moleculesrepresented by the formula XY, wherein X is a hydrophilic moiety and Yis a hydrophobic moiety. In aqueous suspension, the concentric layersare arranged such that the hydrophilic moieties tend to remain incontact with an aqueous phase and the hydrophobic regions tend toself-associate. For example, when aqueous phases are present both withinand without the liposome, the lipid molecules may form a bilayer, knownas a lamella, of the arrangement XY-YX. Aggregates of lipids may formwhen the hydrophilic and hydrophobic parts of more than one lipidmolecule become associated with each other. The size and shape of theseaggregates will depend upon many different variables, such as the natureof the solvent and the presence of other compounds in the solution.

The production of lipid formulations often is accomplished by sonicationor serial extrusion of liposomal mixtures after (I) reverse phaseevaporation (II) dehydration-rehydration (III) detergent dialysis and(IV) thin film hydration. In one aspect, a contemplated method forpreparing liposomes in certain embodiments is heating sonicating, andsequential extrusion of the lipids through filters or membranes ofdecreasing pore size, thereby resulting in the formation of small,stable liposome structures. This preparation producesliposomal/therapeutic compound or liposomes only of appropriate anduniform size, which are structurally stable and produce maximalactivity. Such techniques are well-known to those of skill in the art(see, for example Martin, 1990).

Once manufactured, lipid structures can be used to encapsulate compoundsthat are toxic (e.g., chemotherapeutics) or labile (e.g., nucleic acids)when in circulation. Liposomal encapsulation has resulted in a lowertoxicity and a longer serum half-life for such compounds (Gabizon etal., 1990).

Numerous disease treatments are using lipid based gene transferstrategies to enhance conventional or establish novel therapies, inparticular therapies for treating hyperproliferative diseases. Advancesin liposome formulations have improved the efficiency of gene transferin vivo (Templeton et al., 1997) and it is contemplated that liposomesare prepared by these methods. Alternate methods of preparinglipid-based formulations for nucleic acid delivery are described (WO99/18933).

In another liposome formulation, an amphipathic vehicle called a solventdilution microcarrier (SDMC) enables integration of particular moleculesinto the bi-layer of the lipid vehicle (U.S. Pat. No. 5,879,703). TheSDMCs can be used to deliver lipopolysaccharides, polypeptides, nucleicacids and the like. Of course, any other methods of liposome preparationcan be used by the skilled artisan to obtain a desired liposomeformulation in the present invention.

b. Liposome Targeting

Association of the compositions of the invention with a liposome mayimprove its biodistribution and other properties. For example,liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful (Nicolau and Sene, 1982; Fraley et al.,1979; Nicolau et al., 1987). The feasibility of liposome-mediateddelivery and expression of foreign DNA in cultured chick embryo, HeLaand hepatoma cells has also been demonstrated (Wong et al., 1980).Successful liposome-mediated gene transfer in rats after intravenousinjection has also been accomplished (Nicolau et al., 1987).

It is contemplated that a liposomecomposition may comprise additionalmaterials for delivery to a tissue. For example, in certain embodimentsof the invention, the lipid or liposome may be associated with ahemagglutinating virus (HVJ). This has been shown to facilitate fusionwith the cell membrane and promote cell entry of liposome-encapsulatedDNA (Kaneda et al., 1989). In another example, the lipid or liposome maybe complexed or employed in conjunction with nuclear non-histonechromosomal proteins (HMG-1) (Kato et al., 1991). In yet furtherembodiments, the lipid may be complexed or employed in conjunction withboth HVJ and HMG-1.

Targeted delivery is achieved by the addition of ligands withoutcompromising the ability of these liposomes deliver large amounts of anydisclosed compound of the invention It is contemplated that this willenable delivery to specific cells, tissues and organs. The targetingspecificity of the ligand-based delivery systems are based on thedistribution of the ligand receptors on different cell types. Thetargeting ligand may either be non-covalently or covalently associatedwith the lipid complex, and can be conjugated to the liposomes by avariety of methods.

Exemplary methods for cross-linking ligands (some discussed above) toliposomes are described in U.S. Pat. No. 5,603,872 and U.S. Pat. No.5,401,511, each specifically incorporated herein by reference in itsentirety). Various ligands can be covalently bound to liposomal surfacesthrough the cross-linking of amine residues. Liposomes, in particular,multilamellar vesicles (MLV) or unilamellar vesicles such asmicroemulsified liposomes (MEL) and large unilamellar liposomes (LUVET),each containing phosphatidylethanolamine (PE), have been prepared byestablished procedures. The inclusion of PE in the liposome provides anactive functional residue, a primary amine, on the liposomal surface forcross-linking purposes. Ligands such as epidermal growth factor (EGF)have been successfully linked with PE-liposomes. Ligands are boundcovalently to discrete sites on the liposome surfaces. The number andsurface density of these sites will be dictated by the liposomeformulation and the liposome type. The liposomal surfaces may also havesites for non-covalent association. To form covalent conjugates ofligands and liposomes, cross-linking reagents have been studied foreffectiveness and biocompatibility. Cross-linking reagents includeglutaraldehyde (GAD), bifunctional oxirane (OXR), ethylene glycoldiglycidyl ether (EGDE), and a water soluble carbodjimide, preferably1-ethyl-3-(3-dimethylaminopropyl)carbodjimide (EDC). Through the complexchemistry of cross-linking, linkage of the amine residues of therecognizing substance and liposomes is established.

i. Targeting Ligands

The targeting ligand can be either anchored in the hydrophobic portionof the complex or attached to reactive terminal groups of thehydrophilic portion of the complex. The targeting ligand can be attachedto the liposome via a linkage to a reactive group, e.g., on the distalend of the hydrophilic polymer. Preferred reactive groups include aminogroups, carboxylic groups, hydrazide groups, and thiol groups. Thecoupling of the targeting ligand to the hydrophilic polymer can beperformed by standard methods of organic chemistry that are known tothose skilled in the art. In certain embodiments, the totalconcentration of the targeting ligand can be from about 0.01 to about10% mol.

Targeting ligands are any ligand specific for a characteristic componentof the targeted region. Preferred targeting ligands include proteinssuch as polyclonal or monoclonal antibodies, antibody fragments, orchimeric antibodies, enzymes, or hormones, or sugars such as mono-,oligo- and poly-saccharides (see, Heath et al., (1986)) For example,disialoganglioside GD2 is a tumor antigen that has been identifiedneuroectodermal origin tumors, such as neuroblastoma, melanoma,small-cell lung carcenoma, glioma and certain sarcomas (Cheresh et al.,1986, Schulz et al., 1984). Liposomes containing anti-disialogangliosideGD2 monoclonal antibodies have been used to aid the targeting of theliposomes to cells expressing the tumor antigen (Montaldo et al., 1999;Pagnan et al., 1999). In another non-limiting example, breast andgynecological cancer antigen specific antibodies are described in U.S.Pat. No. 5,939,277, incorporated herein by reference. In a furthernon-limiting example, prostate cancer specific antibodies are disclosedin U.S. Pat. No. 6,107,090, incorporated herein by reference. Thus, itis contemplated that the antibodies described herein or as would beknown to one of ordinary skill in the art may be used to target specifictissues and cell types in combination with the compositions and methodsof the present invention. In certain embodiments of the invention,contemplated targeting ligands interact with integrins, proteoglycans,glycoproteins, receptors or transporters. Suitable ligands include anythat are specific for cells of the target organ, or for structures ofthe target organ exposed to the circulation as a result of localpathology, such as tumors.

In certain embodiments of the present invention, in order to enhance thetransduction of cells, to increase transduction of target cells, or tolimit transduction of undesired cells, antibody or cyclic peptidetargeting moieties (ligands) are associated with the lipid complex. Suchmethods are known in the art. For example, liposomes have been describedfurther that specifically target cells of the mammalian central nervoussystem (U.S. Pat. No. 5,786,214, incorporated herein by reference). Theliposomes are composed essentially ofN-glutarylphosphatidylethanolamine, cholesterol and oleic acid, whereina monoclonal antibody specific for neuroglia is conjugated to theliposomes. It is contemplated that a monoclonal antibody or antibodyfragment may be used to target delivery to specific cells, tissues, ororgans in the animal, such as for example, brain, heart, lung, liver,etc.

Still further, a compound may be delivered to a target cell viareceptor-mediated delivery and/or targeting vehicles comprising a lipidor liposome. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis that will be occurringin a target cell. In view of the cell type-specific distribution ofvarious receptors, this delivery method adds another degree ofspecificity to the present invention.

Thus, in certain aspects of the present invention, a ligand will bechosen to correspond to a receptor specifically expressed on the targetcell population. A cell-specific delivery of compounds of the inventionand/or targeting vehicle may comprise a specific binding ligand incombination with a liposome. The compounds to be delivered are housedwithin a liposome and the specific binding ligand is functionallyincorporated into a liposome membrane. The liposome will thusspecifically bind to the receptor(s) of a target cell and deliver thecontents to a cell. Such systems have been shown to be functional usingsystems in which, for example, epidermal growth factor (EGF) is used inthe receptor-mediated delivery of a nucleic acid to cells that exhibitupregulation of the EGF receptor.

In certain embodiments, a receptor-mediated delivery and/or targetingvehicles comprise a cell receptor-specific ligand and a binding agent.Others comprise a cell receptor-specific ligand to which modfied proteinor a polynucleotide encoding a modified protein to be delivered has beenoperatively attached. For example, several ligands have been used forreceptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al., 1990;Perales et al., 1994; Myers, EPO 0273085), which establishes theoperability of the technique. In another example, specific delivery inthe context of another mammalian cell type has been described (Wu andWu, 1993; incorporated herein by reference).

In still further embodiments, the specific binding ligand may compriseone or more lipids or glycoproteins that direct cell-specific binding.For example, lactosyl-ceramide, a galactose-terminal asialganglioside,have been incorporated into liposomes and observed an increase in theuptake of the insulin gene by hepatocytes (Nicolauetal., 1987). Theasialoglycoprotein, asialofetuin, which contains terminal galactosylresidues, also has been demonstrated to target liposomes to the liver(Spanjer and Scherphof, 1983; Hara et al., 1996). The sugars mannosyl,fucosyl or N-acetyl glucosamine, when coupled to the backbone of apolypeptide, bind the high affinity manose receptor (U.S. Pat. No.5,432,260, specifically incorporated herein by reference in itsentirety). It is contemplated that the cell or tissue-specifictransforming constructs of the present invention can be specificallydelivered into a target cell or tissue in a similar manner.

In another example, lactosyl ceramide, and peptides that target the LDLreceptor related proteins, such as apolipoprotein E3 (“Apo E”) have beenuseful in targeting liposomes to the liver (Spanjer and Scherphof, 1983;WO 98/0748).

Folate and the folate receptor have also been described as useful forcellular targeting (U.S. Pat. No. 5,871,727). In this example, thevitamin folate is coupled to the complex. The folate receptor has highaffinity for its ligand and is overexpressed on the surface of severalmalignant cell lines, including lung, breast and brain tumors.Anti-folate such as methotrexate may also be used as targeting ligands.Transferrin mediated delivery systems target a wide range of replicatingcells that express the transferrin receptor (Gilliland et al., 1980).

C. Liposome/Nucleic Acid Combinations

It is contemplated that when the liposome composition comprises a cellor tissue specific nucleic acid, this technique may have applicabilityin the present invention. In certain embodiments, lipid-based non-viralformulations provide an alternative to viral gene therapies. Althoughmany cell culture studies have documented lipid-based non-viral genetransfer, systemic gene delivery via lipid-based formulations has beenlimited. A major limitation of non-viral lipid-based gene delivery isthe toxicity of the cationic lipids that comprise the non-viral deliveryvehicle. The in vivo toxicity of liposomes partially explains thediscrepancy between in vitro and in vivo gene transfer results. Anotherfactor contributing to this contradictory data is the difference inliposome stability in the presence and absence of serum proteins. Theinteraction between liposomes and serum proteins has a dramatic impacton the stability characteristics of liposomes (Yang and Huang, 1997).Cationic liposomes attract and bind negatively charged serum proteins.Liposomes coated by serum proteins are either dissolved or taken up bymacrophages leading to their removal from circulation. Current in vivoliposomal delivery methods use aerosolization, subcutaneous,intradermal, intratumoral, or intracranial injection to avoid thetoxicity and stability problems associated with cationic lipids in thecirculation. The interaction of liposomes and plasma proteins is largelyresponsible for the disparity between the efficiency of in vitro(Felgner et al., 1987) and in vivo gene transfer (Zhu et al., 1993;Philip et al., 1993; Solodin et al., 1995; Liu et al., 1995; Thierry etal., 1995; Tsukamoto et al., 1995; Aksentijevich et al., 1996).

An exemplary method for targeting viral particles to cells that lack asingle cell-specific marker has been described (U.S. Pat. No.5,849,718). In this method, for example, antibody A may have specificityfor tumor, but also for normal heart and lung tissue, while antibody Bhas specificity for tumor but also normal liver cells. The use ofantibody A or antibody B alone to deliver an anti-proliferative nucleicacid to the tumor would possibly result in unwanted damage to heart andlung or liver cells. However, antibody A and antibody B can be usedtogether for improved cell targeting. Thus, antibody A is coupled to agene encoding an anti-proliferative nucleic acid and is delivered, via areceptor mediated uptake system, to tumor as well as heart and lungtissue. However, the gene is not transcribed in these cells as they lacka necessary transcription factor. Antibody B is coupled to a universallyactive gene encoding the transcription factor necessary for thetranscription of the anti-proliferative nucleic acid and is delivered totumor and liver cells. Therefore, in heart and lung cells only theinactive anti-proliferative nucleic acid is delivered, where it is nottranscribed, leading to no adverse effects. In liver cells, the geneencoding the transcription factor is delivered and transcribed, but hasno effect because no an anti-proliferative nucleic acid gene is present.In tumor cells, however, both genes are delivered and the transcriptionfactor can activate transcription of the anti-proliferative nucleicacid, leading to tumor-specific toxic effects.

The addition of targeting ligands for gene delivery for the treatment ofhyperproliferative diseases permits the delivery of genes whose geneproducts are more toxic than do non-targeted systems. Examples of themore toxic genes that can be delivered includes pro-apoptotic genes suchas Bax and Bak plus genes derived from viruses and other pathogens suchas the adenoviral E4orf4 and the E.coli purine nucleoside phosphorylase,a so-called “suicide gene” which converts the prodrug 6-methylpurinedeoxyriboside to toxic purine 6-methylpurine. Other examples of suicidegenes used with prodrug therapy are the E. coli cytosine deaminase geneand the HSV thymidine kinase gene.

It is also possible to utilize untargeted or targeted lipid complexes togenerate recombinant or modified viruses in vivo. For example, two ormore plasmids could be used to introduce retroviral sequences plus atherapeutic gene into a hyperproliferative cell. Retroviral proteinsprovided in trans from one of the plasmids would permit packaging of thesecond, therapeutic gene-carrying plasmid. Transduced cells, therefore,would become a site for production of non-replicative retrovirusescarrying the therapeutic gene. These retroviruses would then be capableof infecting nearby cells. The promoter for the therapeutic gene may ormay not be inducible or tissue specific.

Similarly, the transferred nucleic acid may represent the DNA for areplication competent or conditionally replicating viral genome, such asan adenoviral genome that lacks all or part of the adenoviral E1a or E2bregion or that has one or more tissue-specific or inducible promotersdriving transcription from the E1a and/or E1b regions. This replicatingor conditional replicating nucleic acid may or may not contain anadditional therapeutic gene such as a tumor suppressor gene oranti-oncogene.

d. Lipid Administration

The actual dosage amount of a lipid composition (e.g., aliposome-modified protein or polynucleotide encoding a modified protein)administered to a patient can be determined by physical andphysiological factors such as body weight, severity of condition,idiopathy of the patient and on the route of administration. With theseconsiderations in mind, the dosage of a lipid composition for aparticular subject and/or course of treatment can readily be determined.

V. EXAMPLES

The following examples are included to demonstrate particularembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutemodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Sequential Deletion Studies

The nucleotide sequence encoding recombinant gelonin (SEQ ID NO:2) wasutilized as the template to create these toxins. Sequence analysis andstructural modeling of rGel showed a significant folding of the moleculeinto pleated sheets, beta coils, and hairpin loops as shown. Accordingto these studies, amino acids 200-277 (C-terminal) appear to fold into abinding pocket similar to that of the docking port of RTA for its Bchain. Since rGel has no B chain, this “docking port” was theorized tobe a vestigial portion of the toxin and may be unnecessary to thebiological activity of this protein.

Sequential deletion mutants of the cDNA molecule encoding rGel from theC-terminal and from the N-terminal were created as shown in FIG. 3A and3B and Table 7 and designated CFR1901-CFR1905. In preliminary studies,constructs CFR 1904, 1905, 2001, 2007, and 2024 demonstrated detectableactivity in Rabbit Reticulocyte Lysate Assays (RRLA). These constructswere 10⁵-10³ less active than CFR 1888, which is considered within arange of“active” toxin molecules (Munishkin et al., 1995).

CFR 1901

To create CFR 1901, which has amino acids 1-46 deleted, 1 μg of purifiedcDNA of CFR 1888 contained in the pX2 vector (originally based onpET-22b, Novagen, Inc.) was digested with 50 units of restrictionendonucleases NcoI and SmaI (Boeringer-Mannheim). The overhang fragmenton the 5′NcoI site was then blunted by addition of 1 unit of Mung Beannuclease(New England Biolabs) and incubated at 30° C. for 0.5 hr. QiagenPCR purification kit was used used to remove the Mung Bean nuclease. Theresulting DNA was circularized by ligation with the 3′ blunted SmaIsite.

CFR 1902

CFR 1888 was digested with Nco I and Cla I restriction endonucleases.The ends were filled in to maintain the correct reading frame, usingKlenow enzyme(New England Biolabs) on the 3′ overhang in order to makeit blunted before religation.

For protein expression, 50 ng of plasmid DNA was transformed into 50 μlof BL21(DE3)pLysS competent E. Coli host cells (Novagen, Inc.).Individual colonies were picked and grow at 37° C. with shaking in 100ml of Luria Broth containing 200 μg/ml of Ampicillin (Sigma ChemicalCo.) up to an OD₆₀₀ between 0.6-0.8. IPTG (Boeringer-Mannheim) was thenadded to the culture to induce the recombinant protein at 0.1 mM finalconcentration. The culture was allowed to incubate for additional 2 hrat 37° C. before harvesting by centrifugation.

TABLE 7 Other Functional Name Designation Description and/or Amino AcidDeletion Amino Acid Replacement/Addition Addition 1888 CFR 1888 noneC-terminus KDPE change to KDEL none KS CFR 1901 CFR 1888 minus AA 1-46none none KC CFR 1902 CFR 1888 minus AA 1-67 none none KB CFR 1903 CFR1888 minus AA 198-251 Additional LAAA before AA 251 none SB CFR 1904 CFR1888 minus AA 1-46 and 198-251 Additional LAAA before AA 252 none CB CFR1905 CFR 1888 minus AA 1-67 and 198-251 Additional LAAA before AA 253none 3825 CFR 2018 CFR 1888 minus AA 104-111 AA 43 = K, C; Additional Lafter AA 164 none CFR 2018 CFR 1888 replaced AA 252-257 VDKDPKA none N10CFR 2001 CFR 2018 minus AA 1-9 none none N43 CFR 2007 CFR 2018 minus AA43-70 none none N87 CFR 2015 CFR 2018 minus AA 87-107 none none N4389CFR 2024 CFR 2018 minus AA 43-70 and 89-109 none none N100130 CFR 2005CFR 2018 minus AA 100-109 and 130-155 none none C211 CFR 2004 CFR 2018minus AA 194-223 and 235-252 none none 3825-Y1 CFR 2019 CFR 2018 minusAA 202-252 FQMVTIDQLKPKIALLKFVK none 3825-Y2 CFR 2020 CFR 2018 replacedAA 28-43 NQWDGTQHGVELRQQ none 3825-Y3 CFR 2021 CFR 2018 replaced AA73-89 IYIMGTQERNEKLFYR none 3825-Y4 CFR 2022 CFR 2018 replaced AA187-196 EENETTCYMG none 4389-Y1 CFR 2025 CFR 2024 replaced AA 153-203FQMVTIDQLKPKIALLKFVK none 4389-Y2 CFR 2026 CFR 2024 replaced AA 28-42NQWDGTQHGVELRQQ none 4389-Y3 CFR 2027 CFR 2024 replaced AA 45-60IYIMGTQERNEKLFYR none 4389-Y4 CFR 2028 CFR 2024 replaced AA 138-147EENETTCYMG none 3825-Y1.2 CFR 2029 CFR 2019 replaced AA 28-43NQWDGTQHGVELRQQ none 3825-Y1.3 CFR 2030 CFR 2019 replaced AA 73-89IYIMGTQERNEKLFYR none 3825-Y1.4 CFR 2031 CFR 2019 replaced AA 187-196EENETTCYMG none 3825-Y2.3 CFR 2032 CFR 2020 replaced AA 73-89IYIMGTQERNEKLFYR none 3825-Y2.4 CFR 2033 CFR 2020 replaced AA 187-196EENETTCYMG none 3825-Y2.3.4 CFR 2034 CFR 2032 replaced AA 187-196EENETTCYMG none 3825-Y1.2.3.4 CFR 2035 CFR 2034 replaced AA 202-251FQMVTIDQLKPKIALLKFVK none 4389-Y1.2 CFR 2036 CFR 2025 replaced AA 28-42NQWDGTQHGVELRQQ none 4389-Y1.3 CFR 2037 CFR 2025 replaced AA 45-60IYIMGTQERNEKLFYR none 4389-Y1.4 CFR 2038 CFR 2025 replaced AA 138-147EENETTCYMG none 4389-Y2.3 CFR 2039 CFR 2026 replaced AA 45-60IYIMGTQERNEKLFYR none 4389-Y2.4 CFR 2040 CFR 2026 replaced AA 138-147EENETTCYMG none 4389-Y2.3.4 CFR 2041 CFR 2039 replaced AA 138-147EENETTCYMG none 4389-Y1.2.3.4 CFR 2042 CFR 2041 replaced AA 153-203FQMVTIDQLKPKIALLKFVK none CB-Y1K CFR 2143 CFR 1905 replaced AA 131-137ISLENKWGKLFQMVTIDQLKPKIALLKFVK none SB-Y1K CFR 2144 CFR 1904 replaced AA152-158 ISLENKWGKLFQMVTIDQLKPKIALLKFVK none 3825-Y1K CFR 2145 CFR 2019plus AA at c-terminus DEL none 4389-Y1K CFR 2146 CFR 2025 plus AA atc-terminus DEL none GrB-CB-Y1K CFR 2247 CFR 2143 plus Granzyme B G4Slinker Human Granzyme B GrB-SB-Y1K CFR 2248 CFR 2144 plus Granzyme B G4Slinker Human Granzyme B GrB-3825-Y1K CFR 2249 CFR 2145 plus Granzyme BG4S linker Human Granzyme B GrB-4389-Y1K CFR 2250 CFR 2146 plus GranzymeB G4S linker Human Granzyme B Bax-CB-Y1K CFR 2351 CFR 2143 plus BaxAlpha G4S linker Human Bax (Full Length) Bax-SB-Y1K CFR 2352 CFR 2144plus Bax Alpha G4S linker Human Bax (Full Length) Bax-3825-Y1K CFR 2353CFR 2145 plus Bax Alpha G4S linker Human Bax (Full Length) Bax-4389-Y1KCFR 2354 CFR 2146 plus Bax Alpha G4S linker Human Bax (Full Length)Bax(3 . . . 6)-CB- CFR 2455 CFR 2143 plus Bax (Truncated) G4S linkerHuman Bax Y1K (Domain 3, 4, 5, 6) Bax(3 . . . 6)-SB- CFR 2456 CFR 2144plus Bax (Truncated) G4S linker Human Bax Y1K (Domain 3, 4, 5, 6) Bax(3. . . 6)- CFR 2457 CFR 2145 plus Bax (Truncated) G4S linker Human Bax3825-Y1K (Domain 3, 4, 5, 6) Bax(3 . . . 6)- CFR 2458 CFR 2146 plus Bax(Truncated) G4S linker Human Bax 4389-Y1K (Domain 3, 4, 5, 6)

Example 2 Map Antipenic Linear Peptide Domains

Antigenic domains on the rgel molecule have not been previouslydescribed in the literature. The antigenic domains of the rgel moleculecan depend on either the carbohydrate or the peptide sequences of themolecule. Recombinant rGel produced in bacteria has no proteinglycosylation, and therefore, antibodies directed against rGel shouldrecognize peptide domains on the molecule.

In order to identify antigenic domains on the rGel molecule, humanpolyclonal antibodies were first isolated from the serum of laboratoryworkers occupationally exposed to recombinant gelonin. Serum obtainedfrom three laboratory workers was added to 96-well ELISA plates coatedwith rGel. The plates were then developed using anti-human antibodies toidentify the presence of human anti-gelonin antibodies. Serum from twoof the three workers showed significant antibody titers compared to thatof the control human serum (FIG. 1). Twenty ml of serum from these twoworkers were then obtained and polyclonal human anti-gelonin antibodieswere obtained by affinity chromatography using Affi-gel affinity resincontaining rGel.

Ten peptides spanning the entire length of the rGel molecule weresynthesized and then used to coat 96 well plates. A solution of thehuman anti-gelonin antibodies was added to the plates, allowed to react,and the presence of bound human antibodies adhering to thepeptide-coated wells was assessed using ELISA. As shown in FIG. 2,significant reactivity of the polyclonal antibodies was obtained withpeptides spanning 23-53 (Domain 1), 72-89 (Domain 2), 181-198 (Domain3), and 223-252 (Domain 4). Designer toxins CFR-2001-2024 were designedto delete these antigenic domains specifically-recognized by humanpolyclonal antibodies to gelonin.

Example 3 Replacement of Antigenic Sequences with Human Sequences

Human/plant chimeric molecule were designed utilizing the informationregarding antigenic domains obtained using human anti-gelonin antibodiesabove to identify four specific antigenic domains in the geloninmolecule (amino acids 205-257, 23-42, 71-88 and 189-204). Thesesequences were further analyzed using the GenQuest/BLAST database tosearch for homologies to known human proteins. An additionalconsideration in this study was to not only identify a “human”homologous sequence, but also to align such a sequence in the designedtoxin molecule so that the enzymatic(n-glycosidic) functionality of theresulting hybrid molecule can be preserved.

Four candidate sequences for insertion into the designer toxin wereidentified from the database and which were used as the basis for aminoacid changes (Table 8). Human homologous sequences for Domain 1 showed ahigh homology (40%) to human KELL protein, which is a blood groupprotein with a zinc binding domain. Interestingly, early studies ofnatural gelonin suggest that the molecule can bind zinc (Sperti et al.,1986), but these studies have not been confirmed. Analysis of Domain 4(amino acids 189-204) demonstrated identity (40%) to human CFAH protein,which appears to play a role as a co-factor in human liver function.Domain 2 (amino acids 23-42) homology search showed 44% homology tohuman UTRO protein, which appears to play a role in cytoskeletalanchoring of cellular plasma membranes.

TABLE 8 HUMAN HOMOLOGUE REPLACEMENT Y1 (205-257) (Original)GKLSFQIRTSGANGMFSEAVELERANGKKYYVTAVDQVKPKIALLKFLEKDPE (Humanized)GKL-FQMVT-------------------------IDQLKPKIALLKFVK---- Y2 (23-42)(Original) ELRVKLKPEGNSHGIPLLRKK (Humanized) ELRVKNQWDGTQHGVEL-RQQ Y3(71-88) (Original) SVYVVGYQVRNRSYFFKD (Humanized) SIYIMGIQERNEKLFYR- Y4(189-204) (Original) QRIRPANNTISLENKW (Humanized) QRIREENETTCYMGKW

Designer toxins containing antigenic site modifications and deletionswere further modified in Domain 3 to conform to 100% identity with HumanKELL protein. Amino acids in the designer molecule flanking the KELLsequence were also adjusted for to closely mimic the alignment ofsequences in the full-length KELL protein. The final sequences designedfor replacement of Domain 3 are demonstrated in Table 8. Table 9 showsthe result of Gen Bank sequence homology searches for the full lengthproteins containing human homologous proteins.

The results of our studies clearly demonstrate that utilizingrecombinant gelonin as an initial template, unique deletions can bedesigned, constructed and tested.

TABLE 9 Analysis of Potential Replacement Peptides for IdentifiedGelonin Antigenic Sequences Designated Y-1, Y-2 Y-3 and Y-4 Protein #Description Identities(%) Sequences Y1 Replacement Query= VELERANGKKYYVTAVDQVKPK P33186 RPIG_GELMU Gelonium Multiflorum 100VELERANGKKYYVTAVDQVKPK P23339 RIPS_PHYAM Phytolacca Americana 47LELKNADGTKWIVLRVDEIKP Q03464 RIPA_PHYAM Phytolacca Americana 47LELKNANGSKWIVLRVDDIEP P10297 RIPC_PHYAM Phytolacca Americana 47LELVDASGAKWIVLRVDEIKP P10978 POLX_TOBAC Nicotiana Tabacum 40MEIESMGGNKYFVTFIDDASRK Q05234 VG27_BPML5 Mycobacteriophage L5 50VELEGVNGERFNLTTGDQ P23276 KELL_HUMAN Homo Sapiens 40LEQRRAQGKLFQMVTIDQLK P21278 GB11_MOUSE Mus Musculus 38EFQLSDSAKYYLTDVDRI Y2 Replacement Query = LRVKLKPEGNSHGIPLLRKK P33186RIPG_GELMU Gelonium Multiflorum 100 LRVKLKPEGNSHGIPLLRKK P24475RIP3_GELMU Gelonium Multiflorum 89 LRVKTKPEGNSHGIPSLRK P09053 AVTA_ECOLIEscherichia Coli 60 LKLDALGNQHGIPLV Q06194 FA8_MOUSE Mus Musculus 47LSLRPHGNSHSIGANEK P39138 ARGI_BACSU Bacillus Subtilis 50 LETSPSGNIHGMPLP10305 ENPP_BPT3 Bacteriophage T3 61 LRVRVKPTGTSEG P43254 COP1_ARATHArabidopsis Thaliana 50 KVEGKAQGSSHGLP P46939 UTRO_HUMAN Homo Sapiens 44RVKNQWDGTQHGVELRQQ Y3 Replacement Query = SVYVVGYQVRNRSYFFKD P33186RIPG_GELMU Gelonium Multiflorum 100 SVYVVGYQVRNRSYFFKD P29339 RTP2_MOMBAMomordica Balsamina 61 NVYVVAYRTRDVSYFFKE Q00465 RIPA_LUFCY LuffaCylindrica 50 NVYIMGYLVNSTSYFFNE P24478 RIPS_TRIKI TrichosanthesKirilowii 50 NVYVMGYRAGDTSYFFNE P02879 RICI_RICCO Ricinus Communis 56NAYVVGYRAGNSAYFF P28590 ABRC_ABRPR Abrus Precatorius 44NAYVVAYRAGSQSYFLRD P23368 MAOM_HUMAN Homo Sapiens 37 IYIMGIQERNEKLFYRP36758 VL2_HPV34 Human Papillomavirus 44 SLYVIPRKRKRLSYFFAD QO5143COX1_PROWL Prototheca Wickerhamii 50 MYVVGLDIIDTRAYF Y4 ReplacementQuery = FQQRIRPANNTISLENKW P33186 RIPG_GELMU Gelonium Multiflorum 100FQQRIRPANNTISLENKW P37874 YGXB_BACSU Bacillus Subtilis 41GQEKIPPAHSSVCLLDKW P34652 CALX_CAEEL Caenorhabditis Elegans 31KGKWIRPKISNPAFKGKW P34110 VP35_YEAST Saccharomyces Cerevisiae 39LQQFIPLVESVIVLSLKW Q07009 CAN2_RAT Rattus Norvegicus 33KLIRIRNPWGQVEWTGKW P37329 MODA_ECOLI Escherichia Coli 50QIEAGAPADLFISADQKW P14336 POLG_TBEVM Tick-borne Encephalitis Virus 83VREDVVCYGGAWSLEEKW P08603 CFAH_HUMAN Homo Sapiens 40 GGFRISEENETTCYMGKWP17632 MBHL_RHOGE Rhodocyclus Gelatinosus 29 LVANIRAGDTATANVEKW

Example 4 Designer Gelonin Toxins

The following section describes gelonin toxins that have beenconstructed using the methods described in the previous examples.

Deletion Toxins

CFR 1888

Starting from our original recombinant gelonin template, the C-terminusfrom the original KDPE was modified to KDEL to facilitate theintracellular tracking of the protein to the intracellular ribosomalcompartment.

CFR 1901-1905 (See Table 7 and FIG. 3A)

Using CFR 1888 as a template, a series of sequential deletion mutantsfrom the N and/or the C-terminus was generated to determine the sectionsof the molecule which could be deleted without affecting biologicalactivity. As shown in FIG. 3A, individual deletions 1901, 1902 and 1903were shown to be inactive. However, when single deletions were combined,(CFR1904 and CFR 1905), biological activity was re-established (FIG.3A).

CFR 2018

Using CFR 1888 as a template, the protein was further modified as shownin Table 7 to make a slightly smaller molecule and to add an alanineresidue at the C-terminus to provide improved in vivo stability duringproduction in a bacterial host.

Toxins Based on Antijenicity Studies

Antigenic domains on the molecule were mapped using linear peptidesspanning the gelonin molecule. As shown in FIG. 1, human polyclonalanti-gelonin antisera revealed four distinct antigenic domains on themolecule. Domain 1 spans amino acids 205-257, Domain 2 is composed ofamino acids 23-42, Domain 3 contains amino acids 71-88, and Domain 4consists of amino acids 89-204.

CFR2001-2024

As shown in FIG. 3B, six deletion mutants were created based on theantigenic domains observed. Three proteins showed biological activitywhile three proteins were inactive.

CFR2019-2042

Replacement studies creating human/plant chimeric molecules. The fourantigenic domains were submitted to GenBank for sequence analysis. ASwissprot protein sequence search was conducted looking for humanhomologous sequences based on the four antigenic domains describedabove.

Human Homologous Sequences

Domain 1 (amino acids 205-257) was found to map to a sequence in thehuman blood group protein KELL(P23276). A 40% identity and a 65%positivity was found to a sequence on this protein.

Domain 2 (amino acids 23-42) was found to have 44% identity match withthe human UTRO protein (P46939).

For Domain 3 (amino acids 71-88), this sequence showed a 37% identityand a 68% positivity to the human protein MAOM (P23368). This protein isdescribed in the Table 8.

For Domain Sequence 4, (189-204) a 40% identity was found in the humanprotein CFAH (P08603).

Human chimeric sequences corresponding to the four antigenic domainswere generated from this data (Table 8). These sequences represent humannon-antigenic replacements for the antigenic domains in the plantprotein.

CFR2019-2024

Starting with CFR2019 and 2024 as templates, numerous new designerproteins were generated designated CFR2019-2042 (Table 7). Theseproteins represent replacement with 1, 2, 3 or 4 domains on the moleculewith human chimeric homologs. Several of these Designer Toxins (CFR2018, 2019, 2024 and 2025) were expressed in bacteria containing a 50kDa tag and purified to homogeneity. Western analysis was performedusing polyclonal antisera to the tag.

The 2019, 2024 and 2025 molecules were reduced in size compared to thestarting template 2018 protein. Western analysis also demonstratesapproximately equivalent reactivity to the anti-tag antibodies showinguniform loading of each toxin molecule on the SDS-PAGE. The Western blotwas re-probed using antibodies to the native CFR 1888 molecule. Therewas good reactivity to the 2018 protein, as shown by Western blot,however, there was virtually no reactivity of this polyclonal antiserato the 2019 and 2025 designer toxins and only slight reactivity to the2024 designer toxin. This indicates that by specific deletion (CFR2024)or replacement of antigenic domains (CFR2019), or a combination ofdeletion and replacement of antigenic domains (CFR2025), new toxinmolecules can be created that are rendered virtually unrecognizable byantibodies to the parent molecule and thus should have a reducedantigenic profile.

CFR 2143-2146

This series of hybrid molecules was designed to incorporate optimalfunctional qualities of the proteins CFR1888 and CFR 2018.

CFR 2247-2458

A series of molecules will be developed combining both the n-glycosidicfunctions of the Type I toxins with those of selected pro-apoptotichuman molecules such as BAX and Granzyme B. These molecules will beassayed for the functional activity of the gelonin component and for theactivities of BAX and Granzyme B. They will also be evaluated forinhibition of cell-free protein synthesis.

An evaluation may first be made on this series of molecules about theexpression of BAX in cells. This can be done using BAX antibodies, suchas the anti-universal Bax 6A7, in immunoassays, such asimmunoprecipitations or Western blotting. After Bax expression isconfirmed, cells will be measured for cell viability. This can be doneby a number of ways, including using a firefly luciferase construct. Todo this a mammalian expression vector pGL3 (Promega) carrying thefirefly luciferase (Luc) structural gene can be transfected into amammalian cell line along with plasmids encoding BAX and BAX fusionproteins. Luciferase activity can be measured by liquid scintillationcounting using 20 ml of the cellular extract. Cell viability will bemeasured as the relative luciferase activity of the tested constructcompared with the specified control plasmid.

Hybrids that include all or part of a granzyme B polypeptide will beevaluated for their enzymatic activity using a fluorimetric measurementof 2-naphthylamine after hydrolysis of L-glutamyl-2-naphthylamide(Bachem, Philadelphia, Pa.). Amidase activity will be measured at 21° C.with 1.00 mM L-glutamyl-2-naphthylamide in buffer A (0.3M NaCl 0.1MHEPES, adjusted to pH 7.0 with 1M NaOH, 1 mM Na₂ EDTA 0.05M (v/v) Tritonx-100) on a Perkin-Elmer 650-10M spectrofluorimeter with fluorescenceexcitation at 340 nm and fluorescence emission observed at 415 nm (bothwith 5 nm bandpass). Small aliquots of enzyme solutions will be added tothe substrate solution, and the fluorescence emission increase will bemonitored for 10-40 min. Alternatively, granzyme B activity will bedetermined in a continuous colorimetric assay, with BAADT(N-a-t-butoxycarbonyl-L-alanyl-L-alanyl-L-aspartyl-thiobenzyl ester) assubstrate. For analysis of column fractions, 1-50 μl will be added tobuffer A with 1 M (v/v) 10 mM BAADT in (CH₃) ₂50 and 1 M (v/v) 11 nMdithiobis (2-nitrobenzoic acid) (Sigma) in CH₃)₂50 at 21° C., and therate of absorbance increase will be measured at 405 nm on a Thermomaxplate reader (Molecular Devices Inc., Palo Alto, Calif.). Absorbanceincreases will be converted to enzymatic rates.

Example 4 Materials and Methods for Example 5

Materials

The cDNA encoding antibody ZME-018 was amplified from hybridoma RNAobtained from hybridoma cells expressing the murine antibody using kitsfrom Novagen (Madison, Wis.) and Invitrogen Corp. (Carlsbad, Calif.).The PCR reagents were obtained from Fisher Scientific (Pittsburgh, Pa.),and the molecular biology enzymes were purchased from either BoehringerMannheim (Indianapolis, Ind.) or New England Biolabs (Beverly, Mass.).Bacterial strains and pEt bacterial expression plasmids were obtainedfrom Novagen (Madison, Wis.) and growth media was purchased from DifcoLaboratories (Detroit, Mich.). All other chemicals and reagents wereeither from Fisher Scientific or Sigma Chemical Co. (St. Louis, Mo.).Metal affinity resin (Talon) was obtained from Clontech Laboratories(Palo Alto, Calif.). Other chromatography resins and materials were fromPharmacia Biotech (Piscataway, N.J.). Tissue culture reagents were fromGIBCO BRL (Gaithersburg, Md.).

Cloning of the VH and VL Domains of Antibody ZME-018

Messenger RNA from murine hybridoma FMT 112 P2 expressing antibodyZME-018 (IgG2A) was isolated using the Invitrogen Fast Track kit andtranscribed to cDNA with the Invitrogen Copy Kit using the specifiedconditions. Amplification of antibody light and heavy chain variableregions was carried out using the Novagen Ig-Prime kit with the mouseIg-primer set. The PCR profile for light-chain amplification was asfollows: 30 cycles of 94° C.×1 min, 60° C.×1 min, and 72° C.×1 minterminated by a 5 min incubation at 72° C. For heavy-chain reactions,the identical conditions were used except that the annealing temperaturewas 50° C. instead of 60° C. DNA amplified using this procedure was thencloned into the Invitrogen T/A cloning vector pCR II without furtherpurification, transformed into E. coli XL1-Blue, and identified usingblue-white screening procedures. Positive clones (five each from theheavy-and light-chain libraries) were sequenced using the T7 and SP6promoter primers and antibody domains identified by homology with otherimmunoglobulin sequences.

Construction of Genes Encoding the Single-chain Antibody scfvMEL and theImmunotoxin scfvMEL/rGel

A two-step splice-overlap extension PCR method (Sambrook et al., 1989)was used to construct the single-chain antibody ZME-018 using light-andheavy-chain DNA clones as templates. Light-chain sequences wereamplified using the primers A (5′-GCTGCCCAACCAGCC ATGGCGGACATTGTGATG-3′)and C (5′-GCCGGAGCCTGGCTTGC(A/C)GCTGCCGCTGGTGAGCCTTGATC(A/T)CCAG-3′),whereas heavy-chain DNA was amplified with the primers B(5′-AAGCCAGGCTCCGGCGAAGGCAGCACCAAAGG CGAAGTGAAGGTT-3′) and D(5′-GCCACCGCCACCACTAGTTGAGGAGACTGT-3′). The PCR profiles for each set ofreactions were as follows: 30 cycles of 1-min denaturation at 94° C., 1min annealing at 50° C., and a 1 min extension at 72° C., followed by afinal 5-min incubation at 72° C. One-tenth volume of each of thesereactions were combined and used directly in a second PCR with onlyprimers A and D following the same reaction profile as before. The finalproduct was purified using Geneclean II (Bio 101, Vista, Calif.),digested with the restriction enzymes NcoI and SpeI, and cloned into theT7-based plasmid vector pET-22b. The genes encoding scfvMEL andrecombinant gelonin were fused together using the splice-overlapextension PCR method with antibody and gelonin DNA as templates andprimers NbsphZME (5′-GGCGGTGGCTCCGTCATGACGGACATTGTGATGACCCAGTCTCAAAAATTC-3′), primer NTXOM (5′-GGTGGCGGTGGCTCCGGTCTAGACACCGTGACG-3′), and primer XOMBAC (5′-AAGGCTCGTGTCGACCTCG AGTCATTAAGCTTTAGGATCTTTATC-3′) (FIG. 4). Purified PCR products were then purifiedand digested as before and cloned into the vector pET-32a. Sequenced DNAclones were subsequently transformed into E. coli strain AD494(DE3) pLysS obtained from Novogen for expression of the fusion toxin.

Protein Expression in E. coli

To express the immunotoxin, bacterial cultures were incubated at 37° C.in 2×YT growth medium with strong antibiotic selection (200 μ/mlampicillin, 70 μg/ml chloramphenicol, and 15 μg/ml of kanamycin) andgrown until early log phase (A₆₀₀=0.4-0.8). The cultures were thendiluted 1:1 with fresh 2×YT medium containing the same concentrations ofantibiotics, and target protein expression was induced at 23° C. by theaddition of 0.1 mM IPTG for 16-23 h. Induced bacterial cultures werethen centrifuged and stored frozen at −80° C. for later purification.

Immunotoxin/Protein Purification

Frozen bacterial pellets from induced cultures expressing immunotoxinscFvZME-Gel were thawed at room temperature and lysed by the addition of1 mg/ml lysozyme in 10 mM Tris-HCl, pH 8.0 for 30 min at 4° C. Thebacterial lysates were then sonicated three times for 10 sec each with acell disruptor and centrifuged at 14,000 rpm for 30 min at 4° C. Thesupernatant was transferred and saved on ice, and the sonicationprocedure was repeated with the cell pellet. Supernatants from the twolysates were then combined and ultracentrifuged at 40,000 rpm in a SS-34rotor for 45 min at 4° C. The samples containing only soluble proteinwere then filtered (0.22 μm pores), adjusted to 40 mM Tris-HCl with 1MTris-HCl (pH 8.0), and then loaded at room temperature onto a Talonmetal-affinity column pre-equilibrated with the same buffer. Afterloading, the column was washed with 3 column volumes of loading buffer,followed by a 5-column volume wash with 40 mM Tris-HCl pH 8.0, 500 mMNaCl, and 5 mM imidazole. Bound protein was then eluted with 5 columnvolumes of buffer containing 40 mM Tris-HCl (pH 8.0), 500 mM NaCl and100-200 mM imidazole. Fractions containing immunotoxin were combined,quantitated, and dialyzed into 20 mM Tris-HCl (pH 7.2), 50 mM NaCl priorto digestion with enterokinase to remove the 6×His tag using theprocedure established by Novagen (Madison, Wis.).

ELISA and Western Analyses

All ELISA incubation steps were at room temperature for 1 h, unlessotherwise specified, and between incubations all wells were washed withELISA wash buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.2% Tween-20).Wells of a 96-well microtiter plate were each coated with 50,000gp240-antigen-positive A375M melanoma cells and dried. These were thenrehydrated and blocked with 3% BSA in wash buffer. Plates wereincubated, and the purified immunotoxin samples, rabbit anti-geloninpolyclonal antibody (at 100 ng/ml in dilution buffer [ELISA wash buffercontaining BSA at a concentration of 1 mg/ml]), andperoxidase-conjugated goat anti-rabbit IgG (Sigma, used at a 1:5,000dilution in dilution buffer) were added. Individual wells werethoroughly washed with wash buffer, and then developed for 30 min. withABTS (2,2′-azino-bis[3-ethylbenz-thiazoline-6-sulfonic acid]) in 0.1 Mcitrate buffer (pH 4.2) and the signal measured at 405 nm.

For Western blots, all incubations were performed at room temperaturefor 1 h, unless otherwise specified. Briefly, proteins were separated bySDS-PAGE and transferred onto nitrocellulose overnight at 4° C. intransfer buffer (25 mM Tris-HCl (pH 7.5), 190 mM glycine, 20% (v/v)HPLC-grade methanol) at 40 v. The filters were blocked with 5% BSA inWestern blocking buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl) and thenreacted successively with rabbit anti-gelonin polyclonal antibody (at aconcentration of 100 ng/mL in Western wash buffer TBS, pH 7.6, 0.5%Tween-20) and peroxidase-conjugated goat anti-rabbit IgG (Sigma, at adilution of 1:10,000 in wash buffer). The signal was developed using theAmersham ECL detection system.

Reticulocyte Lysate in Vitro Translation Assay

The gelonin-induced inhibition of radiolabeled (³H) leucineincorporation into protein in a cell-free protein synthesizing systemfollowing the administration of various doses of immunotoxin was carriedout as specified by the manufacturer (Promega) and as describedpreviously (Press et al., 1986).

Immunofluorescence Staining

Antigen-positive (A375 melanoma) cells were added to polylysine-coated16-well chamber slides (Nunc) at 10⁴ cells per chamber and incubated at37° C. overnight under 5% CO² atmosphere. Cells were treated with aconcentration of 50 υg/ml of the scfvMEL/rGel fusion construct atvarious times. Cells were then washed briefly with PBS, and thenproteins bound to the cell surface were stripped by incubation of 10 minwith glycine buffer (500 mM NaCl, 0.1 M glycine, pH 2.5), neutralizedfor 5 min with 0.5 M Tris, pH 7.4, washed briefly with PBS, and thenfixed in 3.7% formaldehyde (Sigma) for 15 min at room temperature,followed by a brief rinse with PBS. Cells were then permeabilized for 10min in PBS containing 0.2% Triton X-100m, washed three times with PBS,and then incubated with PBS containing 3% BSA for 1 h at roomtemperature. After a brief wash with PBS, cells were incubated witheither rabbit anti-scFvMEL or rabbit anti-rGel polyclonal antibodiesdiluted 1:500 in PBS containing 0.1% Tween-20 and 0.2% BSA for 1 h atroom temperature. Cells were washed three times in PBS containing 0.1%Tween-20 (PBST) for 10 min, blocked for 1 h at room temperature with PBScontaining 3% BSA, followed by 1:100 diluted fluorescein isothiocyanate(FITC)-coupled anti-rabbit IgG (Sigma). Control cells were onlyincubated with the secondary FITC-coupled anti-rabbit IgG (1:100). Afterthree final washes with PBST, cells were washed once in PBS for 10 minand mounted in mounting medium. Slides were analyzed with a fluorescencemicroscope, and each photograph was representative of at least 10 fieldsfor each experiment at 400× magnification.

In Vitro Cytotoxicity Assay

Samples were assayed using a standard 72-h cell proliferation assay withlog-phase (5,000/well) antigen-positive A375M and antigen-negativeMe-180 or SK-OV-3 cell monolayers and using crystal violet stainingprocedures as previously described (Nishikawa et al., 1992).

In Vivo Cytotoxicity Studies

Athymic (nude) mice 4-6 weeks old were divided into groups of 5 mice percage. Log-phase A-375 human melanoma cells (5×10⁶ cells/mouse) wereinjected subcutaneously in the right flank and tumors were allowed toestablish. Once tumors were measurable (˜30-50 mm²), animals weretreated (i.v. tail vein) with either saline (control) or variousconcentrations of the scfvMel/rGel fusion toxin for 4 consecutive days.Animals were monitored and tumors measured for an additional 30 days.

Example 5 Single-chain Recombinant Anti-melanoma Antibody Fused toGelonin Design of scFvMEL/rGel Fusion Protein

The variable region genes for the ZME-018 antibody and the gelonin gene(Rosenblum et al., 1999) were the templates for the construction of theanti-melanoma immunotoxin gene. As a first step, we assembled theimmunotoxin in one orientation and assessed its binding and cytotoxicityto antigen-positive A375M melanoma cells. The genes encoding theantibody and gelonin fragments were linked together using a PCR-basedmethod to construct a fusion in the antibody-gelonin orientations. Theimmunotoxin gene was also C-terminal tagged with a hexahistidinesequence and expressed in E. coli AD494(DE3) pLysS using the NovagenT-7-based expression vector pET-32b.

FIG. 4 illustrates the orientation of the immunotoxin expressed and alsoshows the sequences of amino acid linkers at the junctions of theprotein domains. The antibody was constructed to encode the light chainvariable region (V_(L)) at the N-terminus of the protein with an 18amino acid flexible peptide linker (Alfthan et al., 1995) with the V_(H)C-terminus. Gelonin (CFR2018) (referred to as rgel in this Example) waspositioned downstream of the V_(H) following another linker. We chosethis configuration for reasons involving the unhindered flexibility ofthe antibody-binding site. With the toxin at the N-terminus of thefusion protein, a longer peptide would have been required to provide foroptimal spatial orientation of the two protein moieties, andconstruction of this variant is in progress. DNA-sequencing studies ofthe final fusion gene (FIG. 5) confirmed the sequence of the finalproduct and that no errors had been introduced using this PCR method. Inaddition, sequencing also confirmed that the target gene was insertedinto the correct reading frame in the pET-32b vector.

The protein synthesis inhibitory activity in cell-free systems of therecombinant fusion toxin compared to that of free recombinant geloninsuggests that there is not significant stearic crowding of the geloninactive-site cleft due to proximity of the antibody fragment in ourdesigned molecule. Also, since there are no protein cleavage siteswithin this fusion construct, the data also suggest that gelonin doesnot necessarily require cleavage from the construct to maintainbiological activity. This is in sharp contrast to studies with ricin Achain (RTA), which requires release from the protein carrier to recoverbiological activity (Kim et al., 1988; O'Hare et al., 1990). This issurprising since gelonin and RTA share identical mechanisms of action(Stirpe et al., 1992), and also share approximately 30% sequencehomology (Rosenblum et al., 1999).

Expression and Purification of Fusion Proteins

The plasmid vector pET-32b containing the fusion gene was transformedinto E. coli AD494(DE3) pLysS, and the target protein was induced by theaddition of IPTG. As shown by a coomassie-stained gel, a protein of theexpected molecular mass (68 kDa) was induced. This protein was purifiedusing IMAC resin, and the eluate was exposed to recombinant enterokinase(EK) to yield the final native fusion construct migrating as one band at56 kDa. The fusion construct was also examined by Western blot usingboth an anti-gelonin antibody and an anti-single-chain antibody. ThesfvMEL/rGel fusion construct migrating at 56 kDa reacted with bothantibodies, thus demonstrating the presence of immunoreactive antibodyand toxin components in the fusion construct. Estimated yields ofsoluble sfvMEL/rGel immunotoxin from the induced bacterial cultures wereapproximately 700 μg/L; however, the yield of final, purified fusiontoxin were approximately 200 μg/L. The primary reason for the reducedyield was found to be an inability of the IMAC to completely capture allof the available soluble target protein. Changes made to the bindingbuffers and conditions as well as changing brands of IMAC capture resindid not improve these results.

ELISA Binding of Immunotoxins

To ensure that the purified fusion protein retained antigen-bindingability, the binding of this material was compared to the binding ofintact IgG ZME-018-gelonin chemical conjugate and IgG ZME-018 in acompetition ELISA-based binding assay (FIG. 6) using intactantigen-positive human melanoma cells as the antigen source. ThescfvMEL/rGel fusion construct was found to retain binding affinitiescomparable with the chemical conjugate. The protein demonstratedspecific and significant ELISA binding activity to target A375M melanomacells with background levels of binding to SK-OV-3 or ME-180 cells.

Cell-Free Protein Synthesis Inhibitory Activity of the sfvMEL FusionToxin

The biological activity of toxins can be severely compromised whenincorporated into fusion constructs. In order to examine then-glycosidic activity of the rGel component of the fusion construct,this material was added to an in vitro protein translation assay using³H-leucine incorporation by isolated rabbit reticulocytes. Inhibitioncurves for the fusion construct and native rGel were compared and theIC₅₀ values for the two molecules were found to be virtually identical(100 pM vs 104 pM, respectively).

Binding and Internalization of scfvMEL/rGel by Immunofluorescence

Immunofluorescent staining was done on A375-M cells treated withscFvMEL/rGel at different times after administration. The internalizedconstruct was detected using either rabbit anti-rgel or rabbitanti-scFvMELantibody followed by FITC-coupled anti-rabbit IgG. The rGelmoiety of scFvMEL/rGel fusion protein was observed primarily in cytosolafter treatment, and the amount of rGel in cytosol increased over time.Moreover, scFvMEL moiety of scFvMEL/rGel was also observed in cytosol.This demonstrates that the fusion construct was capable of efficientcell binding and internalization of the rGel toxin after exposure oflog-phase cells.

In Vitro Cytotoxic Activity of Immunotoxins

The sfvMEL/rGel purified fusion protein and the original ZME/rGelchemical construct were tested for specific cytotoxicity against anantigen-positive (A375M) and an antigen-negative (SK-OV-3) cell line. Asshown in FIG. 7, both the chemically-produced and the fusion constructboth demonstrated IC₅₀ values of approximately 10 nM. In contrast, IC₅₀values for the rGel toxin were approximately 200-fold higher(approximately 2,000 nM). The cytotoxic effects of the immunotoxinsagainst antigen-negative SKOV-3 cells was similar to that of the geloninalone. Co-administration of free ZME antibody with the sfvMEL/rGelimmunotoxin (FIG. 8) as expected showed a modest shift in thedose-response curve, demonstrating a dependence of surface antigenrecognition for the development of cellular toxicity of the fusionconstruct.

Antitumor Activity of sfvMEL/rGel in Xenoiraft Models

Mice bearing well-developed A-375 melanoma xenografts were treated witheither saline (controls), or sfvMEL/rGel at either 2 or 20 mg/kg for 4days. As shown in FIG. 9, tumor size in the control group increased from30 to 150 mm² (500% increase) over the 28 day length of the experiment.In contrast, mice treated with the fusion toxin at 2 mg/kg showed aslight decrease in tumor size followed by an increase to approximately60 mm² (100% increase). Mice treated with the 20 mg/kg dose of fusiontoxin demonstrated a 50% decrease in tumor size during treatmentfollowed by a slow recovery of tumor size back to the original tumorsize over 28 days (no increase in overall growth). There were no obvioustoxic effects of the immunotoxin on mice at these doses, suggesting thatthe maximal tolerated dose (MTD) at this schedule had not been reached.

Example 6 In vitro Cytotoxicity Assay

Cell Culture Methods.

Human melanoma tumor cells A375M were maintained in culture usingminimal essential medium (MEM) supplemented with 10% heat-inactivatedfetal bovine serum plus 100 μM non-essential amino-acids, 2 mML-glutamine, 1 mM sodium pyruvate, vitamins, and antibiotics. Culturedcells were screened routinely and found free of mycoplasma infection.

Cell Proliferation Assay

Cell lines were maintained in culture in complete medium at 37° C. in a5% CO₂-humidified air incubator. For assays with recombinant toxins andimmunotoxins, cultures were washed, cells were detached using versene,and resuspended in complete medium at a density of 25×10³ cell/ml. Twohundred μl aliquots were dispensed into 96-well microtiter plates andthe cells were then allowed to adhere. This results in a sparsely seededpopulation of cells. After 24 hours, the media was replaced with mediacontaining different concentrations of either immunotoxins or gelonin.The cells were incubated for 72 hours and analyzed for relative cellproliferation by crystal violet staining.

Crystal Violet Staining

Cells were washed three times with PBS containing calcium and magnesiumfixed and stained with 20% (v/v) methanol containing 0.5% (w/v) crystalviolet. Bound dye was eluted with 150 μl of Sorensen's citrate buffer(0.1 M sodium citrate, pH 4.2-50% (v/v) ethanol) for 1 hour at roomtemperature. The absorbance was measured at 600 nm using a Bio-Tekmicroplate reader. Relative cell proliferation (RCP) was calculated asfollows:

$\begin{matrix}{{RCP} = {\frac{{Mean}\mspace{14mu}{Absorbance}\mspace{11mu}\left( {{Drug}\mspace{14mu}{Treated}} \right)}{{Mean}\mspace{14mu}{Absorbance}\mspace{11mu}\left( {{Non}\text{-}{drug}\mspace{14mu}{Treated}} \right)} \times 100\mspace{11mu}\%}} & \left\lbrack {{eq}\mspace{20mu} 1} \right\rbrack\end{matrix}$

Samples of purified scfvMEL-CFR2018, scfvMEL-CFR2025 and CFR2018 wereassayed using a standard 72-h cell proliferation assay with log-phase(5,000/well) antigen-positive A375M cell monolayers and using crystalviolet staining procedures as previously described.

Results

FIG. 10 shows the cytotoxicity of each of the recombinant moleculestested on the 72 hr growth of the A-375 human melanoma cell line asdescribed above. As shown, both scfvMEL-CFR2018 and scfvMEL-CFR2025fusion constructs inhibited the growth of melanoma cells in culture. Theconcentration of each agent required to inhibit the growth of cells to50% of control values (I.C.₅₀) was 100 nM. In contrast, cell growthinhibition by the free toxin (CFR2018) occurred at over 800 nMconcentration or almost 8-fold higher compared to the antibody fusionconstructs. Antibody targeting of the CFR2018 toxin to tumor cells byfusion to the scfvMEL antibody increases toxicity by 8-fold. Inaddition, in comparison to the CFR2018 toxin, the Designer Toxindesignated CFR2025 has cytotoxic activity comparable to that of theCFR2018 toxin when they are both delivered to tumor cells with anantibody carrier.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of embodiments, it will be apparent to those ofskill in the art that variations may be applied to the compositions andmethods, and in the steps or in the sequence of steps of the methods,described herein without departing from the concept, spirit, and scopeof the invention. More specifically, it will be apparent that certainagents that are both chemically and physiologically related may besubstituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A process for generating a modified protein that has reducedantigenicity comprising: a) selecting a protein one desires toadminister to a first subject; b) identifying a region of the proteinthat is antigenic in the first subject, said region having beenidentified as recognized by an antibody; and c) generating a modifiedprotein in which the identified region is absent.
 2. The process ofclaim 1, wherein the modified protein is prepared by a method furthercomprising: a) screening a human protein database to identify a lessantigenic region that has homology to the antigenic region of theprotein; b) replacing the antigenic region with all or part of theidentified region that is less antigenic to form the modified protein.3. The process of claim 2, wherein the replacing steps is accomplishedrecombinantly using a nucleic acid molecule encoding the modifiedprotein.
 4. The process of claim 1, wherein the modified protein isgenerated by removing the identified region.
 5. The process of claim 4,wherein the absent antigenic region is replaced with the same number ofamino acid residues that are removed.
 6. The process of claim 1, whereinthe absent region comprises at least 5 amino acid residues.
 7. Theprocess of claim 6, wherein the absent region comprises at least 10amino acid residues.
 8. The process of claim 7, wherein the absentregion comprises at least 15 amino acid residues.
 9. The process ofclaim 8, wherein the absent region comprises at least 20 amino acidresidues.
 10. The process of claim 9, wherein the absent regioncomprises at least 25 amino acid residues.
 11. The process of claim 1,wherein the region is identified by ELISA assay.
 12. The process ofclaim 1, wherein the subject is a mammal.
 13. The process of claim 12,wherein the mammal is a human.
 14. The process of claim 1, whereinidentifying a region of the protein that is antigenic in the firstsubject is carried out using antiserum from either the first subject ofa second subject of the same species as the first subject.
 15. Theprocess of claim 14, wherein the confirming step is carried out usingantiserum from either the first subject of a second subject of the samespecies as the first subject.
 16. The process of claim 1, furthercomprising confirming that the modified protein has reduced antigenicityas compared to the protein.